Anti-sirp alpha antibodies

ABSTRACT

The present invention relates to anti-SIRPα antibodies, as well as use of these antibodies in the treatment of diseases such as cancer and infectious disease.

The present application is a continuation of U.S. patent applicationSer. No. 15/953,201, filed Apr. 13, 2018, now U.S. Pat. No. 10,851,164,which claims the benefit of Netherlands Patent Application No. 2018708,filed Apr. 13, 2017, and of Netherlands Patent Application No. 2019166,filed Jul. 3, 2017, each of which is hereby incorporated by reference inits entirety including all tables, figures, and claims.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application includes an electronically submitted sequence listingin .txt format. The .txt file contains a sequence listing entitled“ABE-0007-CT_SeqListing.txt” created on Nov. 30, 2020, and is 233kilobytes in size. The sequence listing contained in this .txt file ispart of the specification and is hereby incorporated by reference hereinin its entirety.

FIELD OF THE INVENTION

The present invention relates to anti-SIRPα antibodies, as well as useof these antibodies in the treatment of diseases.

BACKGROUND OF THE INVENTION

Signal regulatory protein alpha (SIRPα) is membrane glycoprotein fromthe SIRP family. Members of the SIRP family share certain commonstructural motifs. These include a transmembrane segment and anN-terminal extracellular domain that contains three Ig-like loopsconnected by three pairs of disulfide bonds. The C-terminalintracellular domain, however, differs between SIRP family members.SIRPα has an extended intracellular domain containing four tyrosineresidues that form two immunoreceptor tyrosine-based inhibitory motifs(ITIMs), while SIRPβ1 contains a lysine residue in the transmembranedomain followed by a short intracellular tail lacking ITIMs serving as areceptor for DAP12. Eight SIRPα single nucleotide polymorphisms havebeen identified, with the most prevalent being SIRPαV1 and SIRPαV2(Takenaka et al., Nat. Immunol. 2007, 8:1313-23).

“Eat-me” signals (i.e. “altered self”) are extracellular playersspecifically produced by and displayed on the surface of apoptoticcells, but not healthy cells, and are key to the initiation ofphagocytosis by activating phagocytic receptors and subsequent signalingcascades. Eat-me signals require extracellular trafficking in order tobe displayed on apoptotic cells. A particular category of eat-me signalsis provided by membrane-anchored proteins such as phosphatidylserine(PtdSer) and calreticulin (CRT). Externalized PtdSer binds to itsreceptors on phagocytes to facilitate clearance of apoptotic cells (aprocess known as efferocytosis). Likewise, CRT is upregulated on thesurface of apoptotic cells and binds to LDL-receptor-related protein 1(LRP1) on the phagocyte thereby mediating engulfment.

SIRPα is broadly expressed on phagocytes (e.g., macrophages,granulocytes, and dendritic cells) and acts as an inhibitory receptorthrough its interaction with a transmembrane protein CD47. Thisinteraction mediates a response referred to as the “don't eat me”signal. This interaction negatively regulates effector function ofinnate immune cells such as host cell phagocytosis. As CD47 is oftenpresent on tumor cells, this “don't eat me” signal is thought tocontribute to the resistance of tumors to phagocyte-dependent clearance.Despite the similarities in the extracellular domains of SIRPα andSIRPβ1 functional differences exist among the SIRP family members. Forexample, SIRPβ1 does not bind CD47 at detectable levels and so does notmediate the “don't eat me” signal. Instead, SIRPβ1 is involved in theactivation of myeloid cells.

Disruption of CD47-SIRPα signalling (e.g., by antagonistic monoclonalantibodies that bind to either CD47 or SIRPα) reportedly results inenhanced phagocytosis of both solid and hematopoietic tumor cells,including increased phagocytosis of glioblastoma cells in vitro andsignificant anti-tumor activity in vivo.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides anti-SIRPα antibodies andantigen binding fragments thereof comprising the structural andfunctional features specified below.

In various embodiments, the invention provides an antibody or antigenbinding fragment thereof that binds to human SIRPα comprising one, two,or all three of (i), (ii) and (iii): (i) a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 1 or an amino acidsequence differing from SEQ ID NO: 1 by 1, 2, 3, or more conservativesubstitutions; (ii) a heavy chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 2 or an amino acid sequence differingfrom SEQ ID NO: 2 by 1, 2, 3, or more conservative substitutions; and/or(iii) a heavy chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 3 or an amino acid sequence differing from SEQ IDNO: 3 by 1, 2, 3, or more conservative substitutions.

In various other embodiments, the invention provides an antibody orantigen binding fragment thereof that binds to human SIRPα comprisingone, two, or all three of (i), (ii) and (iii): (i) a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 69or an amino acid sequence differing from SEQ ID NO: 1 by 1, 2, 3, ormore conservative substitutions; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 70 or an amino acidsequence differing from SEQ ID NO: 2 by 1, 2, 3, or more conservativesubstitutions; and/or (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 71 or an amino acidsequence differing from SEQ ID NO: 3 by 1, 2, 3, or more conservativesubstitutions.

In certain embodiments, the antibody or antigen binding fragment thereofcomprises a heavy chain variable region comprising an amino acidsequence selected from the group consisting of:

-   -   SEQ ID NO: 75 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 78 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 80 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 82 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 84 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 86 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 88 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% identical thereto,    -   SEQ ID NO: 102 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% identical thereto,    -   SEQ ID NO: 7 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 10 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 12 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 14 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 16 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 18 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto, and    -   SEQ ID NO: 30 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto.

In various embodiments, the invention also provides an antibody orantigen binding fragment thereof that binds to human SIRPα comprisingone, two, or all three of (i), (ii) and (iii): (i) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4or an amino acid sequence differing from SEQ ID NO: 4 by 1, 2, 3, ormore conservative substitutions; (ii) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5 or an amino acidsequence differing from SEQ ID NO: 5 by 1, 2, 3, or more conservativesubstitutions; and/or (iii) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 6 or an amino acidsequence differing from SEQ ID NO: 6 by 1, 2, 3, or more conservativesubstitutions.

In various otherembodiments, the invention also provides an antibody orantigen binding fragment thereof that binds to human SIRPα comprisingone, two, or all three of (i), (ii) and (iii): (i) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 72or an amino acid sequence differing from SEQ ID NO: 4 by 1, 2, 3, ormore conservative substitutions; (ii) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 73 or an amino acidsequence differing from SEQ ID NO: 5 by 1, 2, 3, or more conservativesubstitutions; and/or (iii) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 74 or an amino acidsequence differing from SEQ ID NO: 6 by 1, 2, 3, or more conservativesubstitutions.

In certain embodiments, the antibody or antigen binding fragment thereofcomprises a light chain variable region comprising an amino acidsequence selected from the group consisting of:

-   -   SEQ ID NO: 76 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 90 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 92 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 94 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 96 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 98 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 100 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 104 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% identical thereto,    -   SEQ ID NO: 8 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 20 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 22 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 24 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 26 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 28 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto, and    -   SEQ ID NO: 32 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto.

In various embodiments, the invention provides an antibody or antigenbinding fragment thereof that binds to human SIRPα comprising:

-   -   (i) a heavy chain variable region CDR1 comprising the amino acid        sequence of SEQ ID NO: 1 or an amino acid sequence differing        from SEQ ID NO: 1 by 1, 2, 3, or more conservative        substitutions; (ii) a heavy chain variable region CDR2        comprising the amino acid sequence of SEQ ID NO: 2 or an amino        acid sequence differing from SEQ ID NO: 2 by 1, 2, 3, or more        conservative substitutions; and/or (iii) a heavy chain variable        region CDR3 comprising the amino acid sequence of SEQ ID NO: 3        or an amino acid sequence differing from SEQ ID NO: 3 by 1, 2,        3, or more conservative substitutions;    -   and    -   (iv) a light chain variable region CDR1 comprising the amino        acid sequence of SEQ ID NO: 4 or an amino acid sequence        differing from SEQ ID NO: 4 by 1, 2, 3, or more conservative        substitutions; (v) a light chain variable region CDR2 comprising        the amino acid sequence of SEQ ID NO: 5 or an amino acid        sequence differing from SEQ ID NO: 5 by 1, 2, 3, or more        conservative substitutions; and/or (vi) a light chain variable        region CDR3 comprising the amino acid sequence of SEQ ID NO: 6        or an amino acid sequence differing from SEQ ID NO: 6 by 1, 2,        3, or more conservative substitutions.

In various other embodiments, the invention provides an antibody orantigen binding fragment thereof that binds to human SIRPα comprising:

-   -   (i) a heavy chain variable region CDR1 comprising the amino acid        sequence of SEQ ID NO: 69 or an amino acid sequence differing        from SEQ ID NO: 1 by 1, 2, 3, or more conservative        substitutions; (ii) a heavy chain variable region CDR2        comprising the amino acid sequence of SEQ ID NO: 70 or an amino        acid sequence differing from SEQ ID NO: 2 by 1, 2, 3, or more        conservative substitutions; and/or (iii) a heavy chain variable        region CDR3 comprising the amino acid sequence of SEQ ID NO: 71        or an amino acid sequence differing from SEQ ID NO: 3 by 1, 2,        3, or more conservative substitutions;    -   and    -   (iv) a light chain variable region CDR1 comprising the amino        acid sequence of SEQ ID NO: 72 or an amino acid sequence        differing from SEQ ID NO: 4 by 1, 2, 3, or more conservative        substitutions; (v) a light chain variable region CDR2 comprising        the amino acid sequence of SEQ ID NO: 73 or an amino acid        sequence differing from SEQ ID NO: 5 by 1, 2, 3, or more        conservative substitutions; and/or (vi) a light chain variable        region CDR3 comprising the amino acid sequence of SEQ ID NO: 6        or an amino acid sequence differing from SEQ ID NO: 74 by 1, 2,        3, or more conservative substitutions.

In still other embodiments, the invention provides an antibody orantigen binding fragment thereof that binds to human SIRPα comprising:

-   a heavy chain variable region comprising an amino acid sequence    selected from the group consisting of:    -   SEQ ID NO: 7 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 10 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 12 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 14 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 16 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 18 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto, and    -   SEQ ID NO: 30 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto;        and-   a light chain variable region comprising an amino acid sequence    selected from the group consisting of:    -   SEQ ID NO: 8 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 20 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 22 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 24 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 26 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto, and    -   SEQ ID NO: 28 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto, and    -   SEQ ID NO: 32 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto.

In still other embodiments, the invention provides an antibody orantigen binding fragment thereof that binds to human SIRPα comprising:

-   -   a heavy chain variable region comprising an amino acid sequence        selected from the group consisting of:        -   SEQ ID NO: 75 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% similar or identical thereto,        -   SEQ ID NO: 78 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% similar or identical thereto,        -   SEQ ID NO: 80 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% similar or identical thereto,        -   SEQ ID NO: 82 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% similar or identical thereto,        -   SEQ ID NO: 84 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% similar or identical thereto,        -   SEQ ID NO: 86 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% similar or identical thereto,        -   SEQ ID NO: 88 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% similar or identical thereto; and        -   SEQ ID NO: 102 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,            and    -   a light chain variable region comprising an amino acid sequence        selected from the group consisting of:        -   SEQ ID NO: 76 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% similar or identical thereto,        -   SEQ ID NO: 90 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% similar or identical thereto,        -   SEQ ID NO: 92 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% similar or identical thereto,        -   SEQ ID NO: 94 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% similar or identical thereto,        -   SEQ ID NO: 96 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% similar or identical thereto,        -   SEQ ID NO: 98 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% similar or identical thereto,        -   SEQ ID NO: 100 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% similar or identical thereto, and        -   SEQ ID NO: 104 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto.

In this context, “sequence similarity” is based on the extent ofidentity combined with the extent of conservative changes. Thepercentage of “sequence similarity” is the percentage of amino acids ornucleotides which is either identical or conservatively changed viz.“sequence similarity”=percent sequence identity)+percent conservativechanges). Thus, for the purpose of this invention “conservative changes”and “identity” are considered to be species of the broader term“similarity”. Thus, whenever the term sequence “similarity” is used itembraces sequence “identity” and “conservative changes”. According tocertain embodiments the conservative changes are disregarded and thepercent sequence similarity refers to percent sequence identity. Incertain embodiments, the changes in a sequence permitted by thereferenced percent sequence identity are all or nearly all conservativechanges; that is, when a sequence is 90% identical, the remaining 10%are all or nearly all conservative changes. The term “nearly all” inthis context refers to at least 75% of the permitted sequence changesare conservative changes, more preferably at least 85%, still morepreferably at least 90%, and most preferably at least 95%. In certainembodiments of antibody heavy and/or light chains, the permittedsequence changes are within the framework regions and not in the CDRs.

Preferably said antibody has a heavy chain according to SEQ ID NO: 7.Further preferably said antibody has a light chain according to SEQ IDNO: 8. More preferably, the heavy chain is chosen from any of SEQ ID NO:10, 12, 14, 16, 18, or 30. More preferably, the light chain is chosenfrom any of SEQ ID NO: 20, 22, 24, 26, 28, or 32.

Alternatively, said antibody has a heavy chain according to SEQ ID NO:75. Further preferably said antibody has a light chain according to SEQID NO: 76. More preferably, the heavy chain is chosen from any of SEQ IDNO: 78, 80, 82, 84, 86, 88 or 102. More preferably, the light chain ischosen from any of SEQ ID NO: 90, 92, 94, 96, 98, 100 or 104.

In any of the above embodiments, the antibody or antigen bindingfragment thereof may be isolated, as that term is defined herein.

In any of the above embodiments, the antibody or antigen bindingfragment thereof is a recombinant antibody, as that term is definedherein.

In any of the above embodiments, the antibody or antigen bindingfragment thereof is a full-length antibody, as that term is definedherein.

Antibodies or antigen binding fragments of the present invention may beobtained from a variety of species. For example, the antibodies of thepresent invention may comprise immunoglobulin sequences which arerabbit, mouse, rat, guinea pig, chicken, goat, sheep, donkey, human,llama or camelid sequences, or combinations of such sequences (so-calledchimeric antibodies). Most preferably, the antibodies or antigen bindingfragments are human or humanized antibodies or antigen bindingfragments.

The term antibody includes antigen-binding portions, i.e., “antigenbinding sites,” (e.g., fragments, subsequences, complementaritydetermining regions (CDRs)) that retain capacity to bind antigen,including (i) a Fab fragment, a monovalent fragment consisting of theV_(L), V_(H), C_(L) and C_(H)1 domains; (ii) a F(ab′)2 fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a Fd fragment consisting of the V_(H)and C_(H)1 domains; (iv) a Fv fragment consisting of the V_(L) and V_(H)domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,(1989) Nature 341:544-546), which consists of a VH domain; and (vi) anisolated complementarity determining region (CDR). Single chainantibodies are also included by reference in the term “antibody.”Preferred therapeutic antibodies are intact IgG antibodies. The term“intact IgG” as used herein is meant as a polypeptide belonging to theclass of antibodies that are substantially encoded by a recognizedimmunoglobulin gamma gene. In humans this class comprises IgG1, IgG2,IgG3, and IgG4. In mice this class comprises IgG1, IgG2a, IgG2b, andIgG3. The known Ig domains in the IgG class of antibodies are V_(H),Cγ1, Cγ2, Cγ3, V_(L), and C_(L).

In any of the above embodiments, the antibody or antigen bindingfragment thereof is a human or humanized antibody comprising two heavychains and two light chains. In one embodiment, the antibody is an IgG.In preferred embodiments, antibody is an IgG1, IgG2, or IgG4, andpreferably a human IgG1, IgG2, or IgG4.

In any of the above-mentioned embodiments, the antibody or antigenbinding fragment thereof of the invention can comprise any of the lightchain variable regions described above and a human kappa or lambda lightchain constant domain and an IgG1, IgG2, or IgG4 heavy chain constantdomain. Exemplary light (kappa) and heavy (IgG2 and IgG4) constantregion sequences which may be used in accordance with the invention arerecited in SEQ ID NOs: 63, 65, 67 (each a nucleotide sequence), 64, 66,and 68 (each a polypeptide sequence).

By way of example only, in various embodiments such antibody or antigenbinding fragment thereof comprises one of the following combinations ofheavy chain sequence/light chain variable region sequences:

-   SEQ ID NO: 10/SEQ ID NO: 20 (referred to herein as hSIRPα.50A.H1L1)-   SEQ ID NO: 10/SEQ ID NO: 22 (referred to herein as hSIRPα.50A.H1L2)-   SEQ ID NO: 10/SEQ ID NO: 24 (referred to herein as hSIRPα.50A.H1L3)-   SEQ ID NO: 10/SEQ ID NO: 26 (referred to herein as hSIRPα.50A.H1L4)-   SEQ ID NO: 10/SEQ ID NO: 28 (referred to herein as hSIRPα.50A.H1L5)-   SEQ ID NO: 12/SEQ ID NO: 20 (referred to herein as hSIRPα.50A.H2L1)-   SEQ ID NO: 12/SEQ ID NO: 22 (referred to herein as hSIRPα.50A.H2L2)-   SEQ ID NO: 12/SEQ ID NO: 24 (referred to herein as hSIRPα.50A.H2L3)-   SEQ ID NO: 12/SEQ ID NO: 26 (referred to herein as hSIRPα.50A.H2L4)-   SEQ ID NO: 12/SEQ ID NO: 28 (referred to herein as hSIRPα.50A.H2L5)-   SEQ ID NO: 14/SEQ ID NO: 20 (referred to herein as hSIRPα.50A.H3L1)-   SEQ ID NO: 14/SEQ ID NO: 22 (referred to herein as hSIRPα.50A.H3L2)-   SEQ ID NO: 14/SEQ ID NO: 24 (referred to herein as hSIRPα.50A.H3L3)-   SEQ ID NO: 14/SEQ ID NO: 26 (referred to herein as hSIRPα.50A.H3L4)-   SEQ ID NO: 14/SEQ ID NO: 28 (referred to herein as hSIRPα.50A.H3L5)-   SEQ ID NO: 16/SEQ ID NO: 20 (referred to herein as hSIRPα.50A.H4L1)-   SEQ ID NO: 16/SEQ ID NO: 22 (referred to herein as hSIRPα.50A.H4L2)-   SEQ ID NO: 16/SEQ ID NO: 24 (referred to herein as hSIRPα.50A.H4L3)-   SEQ ID NO: 16/SEQ ID NO: 26 (referred to herein as hSIRPα.50A.H4L4)-   SEQ ID NO: 16/SEQ ID NO: 28 (referred to herein as hSIRPα.50A.H4L5)-   SEQ ID NO: 18/SEQ ID NO: 20 (referred to herein as hSIRPα.50A.H5L1)-   SEQ ID NO: 18/SEQ ID NO: 22 (referred to herein as hSIRPα.50A.H5L2)-   SEQ ID NO: 18/SEQ ID NO: 24 (referred to herein as hSIRPα.50A.H5L3)-   SEQ ID NO: 18/SEQ ID NO: 26 (referred to herein as hSIRPα.50A.H5L4)-   SEQ ID NO: 18/SEQ ID NO: 28 (referred to herein as hSIRPα.50A.H5L5)-   SEQ ID NO: 78/SEQ ID NO: 90 (referred to herein as hSIRPα.40A.H1L1)-   SEQ ID NO: 78/SEQ ID NO: 92 (referred to herein as hSIRPα.40A.H1L2)-   SEQ ID NO: 78/SEQ ID NO: 94 (referred to herein as hSIRPα.40A.H1L3)-   SEQ ID NO: 78/SEQ ID NO: 96 (referred to herein as hSIRPα.40A.H1L4)-   SEQ ID NO: 78/SEQ ID NO: 98 (referred to herein as hSIRPα.40A.H1L5)-   SEQ ID NO: 78/SEQ ID NO: 100 (referred to herein as hSIRPα.40A.H1L6)-   SEQ ID NO: 80/SEQ ID NO: 90 (referred to herein as hSIRPα.40A.H2L1)-   SEQ ID NO: 80/SEQ ID NO: 92 (referred to herein as hSIRPα.40A.H2L2)-   SEQ ID NO: 80/SEQ ID NO: 94 (referred to herein as hSIRPα.40A.H2L3)-   SEQ ID NO: 80/SEQ ID NO: 96 (referred to herein as hSIRPα.40A.H2L4)-   SEQ ID NO: 80/SEQ ID NO: 98 (referred to herein as hSIRPα.40A.H2L5)-   SEQ ID NO: 80/SEQ ID NO: 100 (referred to herein as hSIRPα.40A.H2L6)-   SEQ ID NO: 82/SEQ ID NO: 90 (referred to herein as hSIRPα.40A.H3L1)-   SEQ ID NO: 82/SEQ ID NO: 92 (referred to herein as hSIRPα.40A.H3L2)-   SEQ ID NO: 82/SEQ ID NO: 94 (referred to herein as hSIRPα.40A.H3L3)-   SEQ ID NO: 82/SEQ ID NO: 96 (referred to herein as hSIRPα.40A.H3L4)-   SEQ ID NO: 82/SEQ ID NO: 98 (referred to herein as hSIRPα.40A.H3L5)-   SEQ ID NO: 82/SEQ ID NO: 100 (referred to herein as hSIRPα.40A.H3L6)-   SEQ ID NO: 84/SEQ ID NO: 90 (referred to herein as hSIRPα.40A.H4L1)-   SEQ ID NO: 84/SEQ ID NO: 92 (referred to herein as hSIRPα.40A.H4L2)-   SEQ ID NO: 84/SEQ ID NO: 94 (referred to herein as hSIRPα.40A.H4L3)-   SEQ ID NO: 84/SEQ ID NO: 96 (referred to herein as hSIRPα.40A.H4L4)-   SEQ ID NO: 84/SEQ ID NO: 98 (referred to herein as hSIRPα.40A.H4L5)-   SEQ ID NO: 84/SEQ ID NO: 100 (referred to herein as hSIRPα.40A.H4L6)-   SEQ ID NO: 86/SEQ ID NO: 90 (referred to herein as hSIRPα.40A.H5L1)-   SEQ ID NO: 86/SEQ ID NO: 92 (referred to herein as hSIRPα.40A.H5L2)-   SEQ ID NO: 86/SEQ ID NO: 94 (referred to herein as hSIRPα.40A.H5L3)-   SEQ ID NO: 86/SEQ ID NO: 96 (referred to herein as hSIRPα.40A.H5L4)-   SEQ ID NO: 86/SEQ ID NO: 98 (referred to herein as hSIRPα.40A.H5L5)-   SEQ ID NO: 86/SEQ ID NO: 100 (referred to herein as hSIRPα.40A.H5L6)-   SEQ ID NO: 88/SEQ ID NO: 90 (referred to herein as hSIRPα.40A.H6L1)-   SEQ ID NO: 88/SEQ ID NO: 92 (referred to herein as hSIRPα.40A.H6L2)-   SEQ ID NO: 88/SEQ ID NO: 94 (referred to herein as hSIRPα.40A.H6L3)-   SEQ ID NO: 88/SEQ ID NO: 96 (referred to herein as hSIRPα.40A.H6L4)-   SEQ ID NO: 88/SEQ ID NO: 98 (referred to herein as hSIRPα.40A.H6L5)-   SEQ ID NO: 88/SEQ ID NO: 100 (referred to herein as hSIRPα.40A.H6L6)    or, in each case, at least 90%, 95%, 97%, 98%, or 99% similar or    identical to a respective SEQ ID NO.

In some preferred embodiments, the antibody or antigen binding fragmentis a humanized antibody that comprises two heavy chains and two lightchains, wherein each heavy chain comprises SEQ ID NO: 10 and each lightchain comprises SEQ ID NO: 20, or, in each case, at least 90%, 95%, 97%,98%, or 99% similar or identical to a respective SEQ ID NO, and mostpreferably each light chain comprises a human kappa light chain or ahuman lambda light chain constant domain; and each heavy chain comprisesa human IgG1, IgG2, or IgG4 constant region.

In other preferred embodiments, the antibody or antigen binding fragmentis a humanized antibody that comprises two heavy chains and two lightchains, wherein each heavy chain comprises SEQ ID NO: 16 and each lightchain comprises SEQ ID NO: 28, or, in each case, at least 90%, 95%, 97%,98%, or 99% similar or identical to a respective SEQ ID NO, and mostpreferably each light chain comprises a human kappa light chain or ahuman lambda light chain constant domain; and each heavy chain comprisesa human IgG1, IgG2, or IgG4 constant region.

In still other preferred embodiments, the antibody or antigen bindingfragment is a humanized antibody that comprises two heavy chains and twolight chains, wherein each heavy chain comprises SEQ ID NO: 18 and eachlight chain comprises SEQ ID NO: 20, or, in each case, at least 90%,95%, 97%, 98%, or 99% similar or identical to a respective SEQ ID NO,and most preferably each light chain comprises a human kappa light chainor a human lambda light chain constant domain; and each heavy chaincomprises a human IgG1, IgG2, or IgG4 constant region.

In some preferred embodiments, the antibody or antigen binding fragmentis a humanized antibody that comprises two heavy chains and two lightchains, wherein each heavy chain comprises SEQ ID NO: 80 and each lightchain comprises SEQ ID NO: 90, or, in each case, at least 90%, 95%, 97%,98%, or 99% similar or identical to a respective SEQ ID NO, and mostpreferably each light chain comprises a human kappa light chain or ahuman lambda light chain constant domain; and each heavy chain comprisesa human IgG1, IgG2, or IgG4 constant region.

In some preferred embodiments, the antibody or antigen binding fragmentis a humanized antibody that comprises two heavy chains and two lightchains, wherein each heavy chain comprises SEQ ID NO: 80 and each lightchain comprises SEQ ID NO: 92, or, in each case, at least 90%, 95%, 97%,98%, or 99% similar or identical to a respective SEQ ID NO, and mostpreferably each light chain comprises a human kappa light chain or ahuman lambda light chain constant domain; and each heavy chain comprisesa human IgG1, IgG2, or IgG4 constant region.

In some preferred embodiments, the antibody or antigen binding fragmentis a humanized antibody that comprises two heavy chains and two lightchains, wherein each heavy chain comprises SEQ ID NO: 80 and each lightchain comprises SEQ ID NO: 96, or, in each case, at least 90%, 95%, 97%,98%, or 99% similar or identical to a respective SEQ ID NO, and mostpreferably each light chain comprises a human kappa light chain or ahuman lambda light chain constant domain; and each heavy chain comprisesa human IgG1, IgG2, or IgG4 constant region.

In one embodiment, the anti-SIRPα antibody of the invention comprises afull length antibody structure having two light chains and two heavychains as recited above, wherein each light chain comprises a humankappa light chain or a human lambda light chain constant domain; andeach heavy chain comprises a human IgG1 constant region.

In one embodiment, the anti-SIRPα antibody of the invention comprises afull length antibody structure having two light chains and two heavychains as recited above, wherein each light chain comprises a humankappa light chain or a human lambda light chain constant domain; andeach heavy chain comprises a human IgG2 constant region.

In one embodiment, the anti-SIRPα antibody of the invention comprises afull-length antibody structure having two light chains and two heavychains as recited above, wherein each light chain comprises a humankappa light chain or a human lambda light chain constant domain; andeach heavy chain comprises a human IgG4 constant region.

In certain embodiments, the antibodies or antigen binding fragments ofthe present invention have one, two, three, four, or more, andpreferably each of, the following functional characteristics:

-   -   binds human SIRPαV1 protein having the sequence of SEQ ID NO: 34        with an EC₅₀<1 nM; and exhibits at least a 100-fold higher EC₅₀        for SIRPαV1(P74A) having the sequence of SEQ ID NO: 62; and        optionally also at least a 100-fold higher EC₅₀ for human SIRPβ1        protein having the sequence of SEQ ID NO: 38 (in each case        wherein the reduced EC₅₀ is relative to the EC₅₀ for human        SIRPαV1 protein having the sequence of SEQ ID NO: 34, and in        each case preferably when measured by cellular ELISA (CELISA) as        described hereinafter;    -   binds to a cell expressing human SIRPαV1 protein with an EC₅₀<10        nM, preferably <5 nM, more preferably <1.5 nM, still more        preferably <1.0 nM, even more preferably <0.5 nM, and most        preferably about 0.3 nM or less;    -   binds to a cell expressing human SIRPαV2 protein with an EC₅₀<10        nM, preferably <5 nM, more preferably <1.5 nM, still more        preferably <1.0 nM, even more preferably <0.5 nM, and most        preferably about 0.3 nM or less;    -   does not appreciably bind to SIRPβ1 protein at an antibody        concentration of 50 nM, preferably 67 nM, and more preferably        100 nM; or alternatively at a concentration that is 10-fold        greater, preferably 50-fold greater, more preferably 100-fold        greater, and still more preferably 200-fold greater than the        antibody's EC₅₀ for SIRPαV1 or SIRPαV2;

inhibits binding between human SIRPα and CD47 with an IC₅₀<10.0 nM, morepreferably <5.0 nM, still more preferably <2.5 nM, and most preferablyabout 1.0 nM or less; and

-   -   exhibits a T20 “humanness” score of at least 79, and more        preferably 85.

Preferably, the anti-SIRPα antibodies or antigen binding fragments ofthe invention do not appreciably bind to one or both of SIRPαV1(P74A)and SIRPβ1 protein at an antibody concentration of 100 nM oralternatively at an antibody concentration that is 200-fold greater thanthe antibody's EC₅₀ for SIRPαV1 or SIRPαV2, while binding to a cellexpressing human SIRPαV1 protein with an EC_(50 <10) nM. Mostpreferably, each light chain comprises a human kappa light chain or ahuman lambda light chain constant domain; and each heavy chain comprisesa human IgG1, IgG2, or IgG4constant region.

In certain embodiments, the anti-SIRPα antibody or antigen bindingfragment thereof of the invention can be conjugated to at least onetherapeutic agent. In one embodiment, the therapeutic agent is a secondantibody or fragment thereof, an immunomodulator, a hormone, a cytotoxicagent, an enzyme, a radionuclide, or a second antibody conjugated to atleast one immunomodulator, enzyme, radioactive label, hormone, antisenseoligonucleotide, or cytotoxic agent, or a combination thereof.

The invention also provides isolated polypeptides comprising the aminoacid sequence of any one of SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 76,90, 92, 94, 96, 98, 100, 102, 104, 7, 10, 12, 14, 16, 18, 30, 8, 20, 22,24, 26, 28, and 32 or a fragment of any said sequences, or an amino acidsequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

The invention also provides isolated nucleic acids encoding anyone ofthe anti-SIRPα antibodies or antigen binding fragments of the invention.

In one embodiment, the invention provides an isolated nucleic acid whichencodes an amino acid sequence selected from the group consisting of:

-   -   SEQ ID NO: 75 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 78 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 80 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 82 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 84 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 86 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 88 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 102 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 10 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 12 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 14 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 16 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 18 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto, and    -   SEQ ID NO: 30 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 10 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 9or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 12 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 11or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 14 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 13or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 16 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 15or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 18 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 17or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 30 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 29or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 78 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 77or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 80 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 79or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 82 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 81or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 84 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 83or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 86 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 85or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 88 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 87or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 102 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO:101 or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%identical thereto.

In one embodiment, the invention provides an isolated nucleic acid whichencodes an amino acid sequence selected from the group consisting of:

-   -   SEQ ID NO: 76 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 90 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 92 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 94 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 96 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 98 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 100 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 104 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 8 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 20 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 22 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 24 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 26 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto,    -   SEQ ID NO: 28 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto, and    -   SEQ ID NO: 32 or an amino acid sequence at least 90%, 95%, 97%,        98%, or 99% similar or identical thereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 20 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 19or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 22 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 21or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 24 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 23or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 26 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 25or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 28 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 27or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 32 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 31or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 90 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 89or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 92 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 91or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 94 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 93or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 96 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 95or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 98 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 97or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 100 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO: 99or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identicalthereto.

In certain embodiments, the amino acid sequence of SEQ ID NO: 104 or anamino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar oridentical thereto is encoded by a nucleic acid sequence of SEQ ID NO:103 or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%identical thereto.

In certain embodiments, the isolated nucleic acids of the presentinvention can optionally comprise a leader sequence.

Such nucleic acids can comprise one or more of the following nucleicacid sequences:

-   -   a nucleic acid sequence of SEQ ID NO: 77 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 79 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 81 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 83 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 85 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 87 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 101 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 89 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 91 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 93 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 95 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 97 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 99 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 101 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 103 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 9 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 15 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 29 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 19 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 21 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 23 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 25 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 27 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,        and/or    -   a nucleic acid sequence of SEQ ID NO: 31 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

In certain embodiments, the nucleic acid can encode a human or humanizedantibody, and includes nucleic acid sequences for both heavy and lightchains. In one embodiment, the antibody is an IgG. In preferredembodiments, antibody is an IgG1, IgG2, or IgG4, and preferably a humanIgG1, IgG2, or IgG4. In certain embodiments, the light chain sequencecomprises a human kappa light chain or a human lambda light chainconstant domain sequence; and each heavy chain sequence comprises ahuman IgG1, IgG2, or IgG4 constant region sequence.

Preferably, such nucleic acids comprise the following combination heavychain and light chain variable region nucleic acid sequences:

-   SEQ ID NO: 9/SEQ ID NO: 19 (referred to herein as hSIRPα.50A.H1L1)-   SEQ ID NO: 9/SEQ ID NO: 21 (referred to herein as hSIRPα.50A.H1L2)-   SEQ ID NO: 9/SEQ ID NO: 23 (referred to herein as hSIRPα.50A.H1L3)-   SEQ ID NO: 9/SEQ ID NO: 25 (referred to herein as hSIRPα.50A.H1L4)-   SEQ ID NO: 9/SEQ ID NO: 27 (referred to herein as hSIRPα.50A.H1L5)-   SEQ ID NO: 11/SEQ ID NO: 19 (referred to herein as hSIRPα.50A.H2L1)-   SEQ ID NO: 11/SEQ ID NO: 21 (referred to herein as hSIRPα.50A.H2L2)-   SEQ ID NO: 11/SEQ ID NO: 23 (referred to herein as hSIRPα.50A.H2L3)-   SEQ ID NO: 11/SEQ ID NO: 25 (referred to herein as hSIRPα.50A.H2L4)-   SEQ ID NO: 11/SEQ ID NO: 27 (referred to herein as hSIRPα.50A.H2L5)-   SEQ ID NO: 13/SEQ ID NO: 19 (referred to herein as hSIRPα.50A.H3L1)-   SEQ ID NO: 13/SEQ ID NO: 21 (referred to herein as hSIRPα.50A.H3L2)-   SEQ ID NO: 13/SEQ ID NO: 23 (referred to herein as hSIRPα.50A.H3L3)-   SEQ ID NO: 13/SEQ ID NO: 25 (referred to herein as hSIRPα.50A.H3L4)-   SEQ ID NO: 13/SEQ ID NO: 27 (referred to herein as hSIRPα.50A.H3L5)-   SEQ ID NO: 15/SEQ ID NO: 19 (referred to herein as hSIRPα.50A.H4L1)-   SEQ ID NO: 15/SEQ ID NO: 21 (referred to herein as hSIRPα.50A.H4L2)-   SEQ ID NO: 15/SEQ ID NO: 23 (referred to herein as hSIRPα.50A.H4L3)-   SEQ ID NO: 15/SEQ ID NO: 25 (referred to herein as hSIRPα.50A.H4L4)-   SEQ ID NO: 15/SEQ ID NO: 27 (referred to herein as hSIRPα.50A.H4L5)-   SEQ ID NO: 17/SEQ ID NO: 19 (referred to herein as hSIRPα.50A.H5L1)-   SEQ ID NO: 17/SEQ ID NO: 21 (referred to herein as hSIRPα.50A.H5L2)-   SEQ ID NO: 17/SEQ ID NO: 23 (referred to herein as hSIRPα.50A.H5L3)-   SEQ ID NO: 17/SEQ ID NO: 25 (referred to herein as hSIRPα.50A.H5L4)-   SEQ ID NO: 17/SEQ ID NO: 27 (referred to herein as hSIRPα.50A.H5L5)-   SEQ ID NO: 77/SEQ ID NO: 89 (referred to herein as hSIRPα.40A.H1L1)-   SEQ ID NO: 77/SEQ ID NO: 91 (referred to herein as hSIRPα.40A.H1L2)-   SEQ ID NO: 77/SEQ ID NO: 93 (referred to herein as hSIRPα.40A.H1L3)-   SEQ ID NO: 77/SEQ ID NO: 95 (referred to herein as hSIRPα.40A.H1L4)-   SEQ ID NO: 77/SEQ ID NO: 97 (referred to herein as hSIRPα.40A.H1L5)-   SEQ ID NO: 77/SEQ ID NO: 99 (referred to herein as hSIRPα.40A.H1L6)-   SEQ ID NO: 79/SEQ ID NO: 89 (referred to herein as hSIRPα.40A.H2L1)-   SEQ ID NO: 79/SEQ ID NO: 91 (referred to herein as hSIRPα.40A.H2L2)-   SEQ ID NO: 79/SEQ ID NO: 93 (referred to herein as hSIRPα.40A.H2L3)-   SEQ ID NO: 79/SEQ ID NO: 95 (referred to herein as hSIRPα.40A.H2L4)-   SEQ ID NO: 79/SEQ ID NO: 97 (referred to herein as hSIRPα.40A.H2L5)-   SEQ ID NO: 79/SEQ ID NO: 99 (referred to herein as hSIRPα.40A.H2L6)-   SEQ ID NO: 81/SEQ ID NO: 89 (referred to herein as hSIRPα.40A.H3L1)-   SEQ ID NO: 81/SEQ ID NO: 91 (referred to herein as hSIRPα.40A.H3L2)-   SEQ ID NO: 81/SEQ ID NO: 93 (referred to herein as hSIRPα.40A.H3L3)-   SEQ ID NO: 81/SEQ ID NO: 95 (referred to herein as hSIRPα.40A.H3L4)-   SEQ ID NO: 81/SEQ ID NO: 97 (referred to herein as hSIRPα.40A.H3L5)-   SEQ ID NO: 81/SEQ ID NO: 99 (referred to herein as hSIRPα.40A.H3L6)-   SEQ ID NO: 83/SEQ ID NO: 89 (referred to herein as hSIRPα.40A.H4L1)-   SEQ ID NO: 83/SEQ ID NO: 91 (referred to herein as hSIRPα.40A.H4L2)-   SEQ ID NO: 83/SEQ ID NO: 93 (referred to herein as hSIRPα.40A.H4L3)-   SEQ ID NO: 83/SEQ ID NO: 95 (referred to herein as hSIRPα.40A.H4L4)-   SEQ ID NO: 83/SEQ ID NO: 97 (referred to herein as hSIRPα.40A.H4L5)-   SEQ ID NO: 83/SEQ ID NO: 99 (referred to herein as hSIRPα.40A.H4L6)-   SEQ ID NO: 85/SEQ ID NO: 89 (referred to herein as hSIRPα.40A.H5L1)-   SEQ ID NO: 85/SEQ ID NO: 91 (referred to herein as hSIRPα.40A.H5L2)-   SEQ ID NO: 85/SEQ ID NO: 93 (referred to herein as hSIRPα.40A.H5L3)-   SEQ ID NO: 85/SEQ ID NO: 95 (referred to herein as hSIRPα.40A.H5L4)-   SEQ ID NO: 85/SEQ ID NO: 97 (referred to herein as hSIRPα.40A.H5L5)-   SEQ ID NO: 85/SEQ ID NO: 99 (referred to herein as hSIRPα.40A.H5L6)-   SEQ ID NO: 87/SEQ ID NO: 89 (referred to herein as hSIRPα.40A.H6L1)-   SEQ ID NO: 87/SEQ ID NO: 91 (referred to herein as hSIRPα.40A.H6L2)-   SEQ ID NO: 87/SEQ ID NO: 93 (referred to herein as hSIRPα.40A.H6L3)-   SEQ ID NO: 87/SEQ ID NO: 95 (referred to herein as hSIRPα.40A.H6L4)-   SEQ ID NO: 87/SEQ ID NO: 97 (referred to herein as hSIRPα.40A.H6L5)-   SEQ ID NO: 87/SEQ ID NO: 99 (referred to herein as hSIRPα.40A.H6L6)    or, in each case, at least 90%, 95%, 97%, 98%, or 99% identical to a    respective SEQ ID NO.

In some preferred embodiments, the nucleic acid comprises SEQ ID NO: 9and SEQ ID NO: 19 or, in each case, at least 90%, 95%, 97%, 98%, or 99%identical to a respective SEQ ID NO.

In some preferred embodiments, the nucleic acid comprises SEQ ID NO: 15and SEQ ID NO: 27 or, in each case, at least 90%, 95%, 97%, 98%, or 99%identical to a respective SEQ ID NO.

In some preferred embodiments, the nucleic acid comprises SEQ ID NO: 17and SEQ ID NO: 19 or, in each case, at least 90%, 95%, 97%, 98%, or 99%identical to a respective SEQ ID NO.

In some preferred embodiments, the nucleic acid comprises SEQ ID NO: 79and SEQ ID NO: 89 or, in each case, at least 90%, 95%, 97%, 98%, or 99%identical to a respective SEQ ID NO.

In some preferred embodiments, the nucleic acid comprises SEQ ID NO: 79and SEQ ID NO: 91 or, in each case, at least 90%, 95%, 97%, 98%, or 99%identical to a respective SEQ ID NO.

In some preferred embodiments, the nucleic acid comprises SEQ ID NO: 79and SEQ ID NO: 95 or, in each case, at least 90%, 95%, 97%, 98%, or 99%identical to a respective SEQ ID NO.

The invention also provides expression vectors comprising one or morenucleic acids of the present invention. An expression vector is a DNAmolecule comprising the regulatory elements necessary for transcriptionof a target nucleic acid in a host cell. Typically, the target nucleicacid is placed under the control of certain regulatory elementsincluding constitutive or inducible promoters, tissue-specificregulatory elements, and enhancer elements. Such a target nucleic acidis said to be “operably linked to” the regulatory elements when theregulating element controls the expression of the gene.

These isolated nucleic acids and the expression vectors comprising themmay be used to express the antibodies of the invention or antigenbinding fragments thereof in recombinant host cells. Thus, the inventionalso provides host cells comprising an expression vector of the presentinvention.

Such expression vectors can comprise one or more of the followingnucleic acid sequences operably linked to regulatory elements:

-   -   a nucleic acid sequence of SEQ ID NO: 77 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 79 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 81 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 83 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 85 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 87 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 101 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 89 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 91 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 93 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 95 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 97 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 99 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 103 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 15 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 29 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 19 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 21 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 23 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 25 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 27 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,        and/or    -   a nucleic acid sequence of SEQ ID NO: 31 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

In certain embodiments, the expression vector comprises nucleic acidsequences encoding both a heavy chain sequence and a light chainsequence of an anti-SIRPα antibody of the present invention. Preferably,such expression vectors comprise the following combination heavy chainand light chain variable region nucleic acid sequences:

-   SEQ ID NO: 9/SEQ ID NO: 19 (referred to herein as hSIRPα.50A.H1L1)-   SEQ ID NO: 9/SEQ ID NO: 21 (referred to herein as hSIRPα.50A.H1L2)-   SEQ ID NO: 9/SEQ ID NO: 23 (referred to herein as hSIRPα.50A.H1L3)-   SEQ ID NO: 9/SEQ ID NO: 25 (referred to herein as hSIRPα.50A.H1L4)-   SEQ ID NO: 9/SEQ ID NO: 27 (referred to herein as hSIRPα.50A.H1L5)-   SEQ ID NO: 11/SEQ ID NO: 19 (referred to herein as hSIRPα.50A.H2L1)-   SEQ ID NO: 11/SEQ ID NO: 21 (referred to herein as hSIRPα.50A.H2L2)-   SEQ ID NO: 11/SEQ ID NO: 23 (referred to herein as hSIRPα.50A.H2L3)-   SEQ ID NO: 11/SEQ ID NO: 25 (referred to herein as hSIRPα.50A.H2L4)-   SEQ ID NO: 11/SEQ ID NO: 27 (referred to herein as hSIRPα.50A.H2L5)-   SEQ ID NO: 13/SEQ ID NO: 19 (referred to herein as hSIRPα.50A.H3L1)-   SEQ ID NO: 13/SEQ ID NO: 21 (referred to herein as hSIRPα.50A.H3L2)-   SEQ ID NO: 13/SEQ ID NO: 23 (referred to herein as hSIRPα.50A.H3L3)-   SEQ ID NO: 13/SEQ ID NO: 25 (referred to herein as hSIRPα.50A.H3L4)-   SEQ ID NO: 13/SEQ ID NO: 27 (referred to herein as hSIRPα.50A.H3L5)-   SEQ ID NO: 15/SEQ ID NO: 19 (referred to herein as hSIRPα.50A.H4L1)-   SEQ ID NO: 15/SEQ ID NO: 21 (referred to herein as hSIRPα.50A.H4L2)-   SEQ ID NO: 15/SEQ ID NO: 23 (referred to herein as hSIRPα.50A.H4L3)-   SEQ ID NO: 15/SEQ ID NO: 25 (referred to herein as hSIRPα.50A.H4L4)-   SEQ ID NO: 15/SEQ ID NO: 27 (referred to herein as hSIRPα.50A.H4L5)-   SEQ ID NO: 17/SEQ ID NO: 19 (referred to herein as hSIRPα.50A.H5L1)-   SEQ ID NO: 17/SEQ ID NO: 21 (referred to herein as hSIRPα.50A.H5L2)-   SEQ ID NO: 17/SEQ ID NO: 23 (referred to herein as hSIRPα.50A.H5L3)-   SEQ ID NO: 17/SEQ ID NO: 25 (referred to herein as hSIRPα.50A.H5L4)-   SEQ ID NO: 17/SEQ ID NO: 27 (referred to herein as hSIRPα.50A.H5L5)-   SEQ ID NO: 77/SEQ ID NO: 89 (referred to herein as hSIRPα.40A.H1L1)-   SEQ ID NO: 77/SEQ ID NO: 91 (referred to herein as hSIRPα.40A.H1L2)-   SEQ ID NO: 77/SEQ ID NO: 93 (referred to herein as hSIRPα.40A.H1L3)-   SEQ ID NO: 77/SEQ ID NO: 95 (referred to herein as hSIRPα.40A.H1L4)-   SEQ ID NO: 77/SEQ ID NO: 97 (referred to herein as hSIRPα.40A.H1L5)-   SEQ ID NO: 77/SEQ ID NO: 99 (referred to herein as hSIRPα.40A.H1L6)-   SEQ ID NO: 79/SEQ ID NO: 89 (referred to herein as hSIRPα.40A.H2L1)-   SEQ ID NO: 79/SEQ ID NO: 91 (referred to herein as hSIRPα.40A.H2L2)-   SEQ ID NO: 79/SEQ ID NO: 93 (referred to herein as hSIRPα.40A.H2L3)-   SEQ ID NO: 79/SEQ ID NO: 95 (referred to herein as hSIRPα.40A.H2L4)-   SEQ ID NO: 79/SEQ ID NO: 97 (referred to herein as hSIRPα.40A.H2L5)-   SEQ ID NO: 79/SEQ ID NO: 99 (referred to herein as hSIRPα.40A.H2L6)-   SEQ ID NO: 81/SEQ ID NO: 89 (referred to herein as hSIRPα.40A.H3L1)-   SEQ ID NO: 81/SEQ ID NO: 91 (referred to herein as hSIRPα.40A.H3L2)-   SEQ ID NO: 81/SEQ ID NO: 93 (referred to herein as hSIRPα.40A.H3L3)-   SEQ ID NO: 81/SEQ ID NO: 95 (referred to herein as hSIRPα.40A.H3L4)-   SEQ ID NO: 81/SEQ ID NO: 97 (referred to herein as hSIRPα.40A.H3L5)-   SEQ ID NO: 81/SEQ ID NO: 99 (referred to herein as hSIRPα.40A.H3L6)-   SEQ ID NO: 83/SEQ ID NO: 89 (referred to herein as hSIRPα.40A.H4L1)-   SEQ ID NO: 83/SEQ ID NO: 91 (referred to herein as hSIRPα.40A.H4L2)-   SEQ ID NO: 83/SEQ ID NO: 93 (referred to herein as hSIRPα.40A.H4L3)-   SEQ ID NO: 83/SEQ ID NO: 95 (referred to herein as hSIRPα.40A.H4L4)-   SEQ ID NO: 83/SEQ ID NO: 97 (referred to herein as hSIRPα.40A.H4L5)-   SEQ ID NO: 83/SEQ ID NO: 99 (referred to herein as hSIRPα.40A.H4L6)-   SEQ ID NO: 85/SEQ ID NO: 89 (referred to herein as hSIRPα.40A.H5L1)-   SEQ ID NO: 85/SEQ ID NO: 91 (referred to herein as hSIRPα.40A.H5L2)-   SEQ ID NO: 85/SEQ ID NO: 93 (referred to herein as hSIRPα.40A.H5L3)-   SEQ ID NO: 85/SEQ ID NO: 95 (referred to herein as hSIRPα.40A.H5L4)-   SEQ ID NO: 85/SEQ ID NO: 97 (referred to herein as hSIRPα.40A.H5L5)-   SEQ ID NO: 85/SEQ ID NO: 99 (referred to herein as hSIRPα.40A.H5L6)-   SEQ ID NO: 87/SEQ ID NO: 89 (referred to herein as hSIRPα.40A.H6L1)-   SEQ ID NO: 87/SEQ ID NO: 91 (referred to herein as hSIRPα.40A.H6L2)-   SEQ ID NO: 87/SEQ ID NO: 93 (referred to herein as hSIRPα.40A.H6L3)-   SEQ ID NO: 87/SEQ ID NO: 95 (referred to herein as hSIRPα.40A.H6L4)-   SEQ ID NO: 87/SEQ ID NO: 97 (referred to herein as hSIRPα.40A.H6L5)-   SEQ ID NO: 87/SEQ ID NO: 99 (referred to herein as hSIRPα.40A.H6L6)    or, in each case, at least 90%, 95%, 97%, 98%, or 99% identical to a    respective SEQ ID NO.

In any of the above embodiments, the expression vector can encode forexpression a human or humanized antibody, and includes nucleic acidsequences for both heavy and light chains. In one embodiment, theantibody is an IgG. In preferred embodiments, antibody is an IgG1, IgG2,or IgG4, and preferably a human IgG1, IgG2, or IgG4. In certainembodiments, the light chain sequence comprises a human kappa lightchain or a human lambda light chain constant domain sequence; and eachheavy chain sequence comprises a human IgG4 constant region sequence.

In some preferred embodiments, the expression vector encodes forexpression a human or humanized antibody, wherein the heavy chainnucleic acid sequence comprises SEQ ID NO: 9 and the light chain nucleicacid sequence comprises SEQ ID NO: 19, or, in each case, at least 90%,95%, 97%, 98%, or 99% identical to a respective SEQ ID NO, and is mostpreferably an IgG1, IgG2, or IgG4 isotype.

In some preferred embodiments, the expression vector encodes forexpression a human or humanized antibody, wherein the heavy chainnucleic acid sequence comprises SEQ ID NO: 15 and the light chainnucleic acid sequence comprises SEQ ID NO: 27, or, in each case, atleast 90%, 95%, 97%, 98%, or 99% identical to a respective SEQ ID NO,and is most preferably an IgG1, IgG2, or IgG4 isotype.

In some preferred embodiments, the expression vector encodes forexpression a human or humanized antibody, wherein the heavy chainnucleic acid sequence comprises SEQ ID NO: 17 and the light chainnucleic acid sequence comprises SEQ ID NO: 19, or, in each case, atleast 90%, 95%, 97%, 98%, or 99% identical to a respective SEQ ID NO or,in each case, at least 90%, 95%, 97%, 98%, or 99% identical to arespective SEQ ID NO, and is most preferably an IgG1, IgG2, or IgG4isotype.

In some preferred embodiments, the expression vector encodes forexpression a human or humanized antibody, wherein the heavy chainnucleic acid sequence comprises SEQ ID NO: 79 and the light chainnucleic acid sequence comprises SEQ ID NO: 89, or, in each case, atleast 90%, 95%, 97%, 98%, or 99% identical to a respective SEQ ID NO,and is most preferably an IgG1, IgG2, or IgG4 isotype.

In some preferred embodiments, the expression vector encodes forexpression a human or humanized antibody, wherein the heavy chainnucleic acid sequence comprises SEQ ID NO: 79 and the light chainnucleic acid sequence comprises SEQ ID NO: 91, or, in each case, atleast 90%, 95%, 97%, 98%, or 99% identical to a respective SEQ ID NO,and is most preferably an IgG1, IgG2, or IgG4 isotype.

In some preferred embodiments, the expression vector encodes forexpression a human or humanized antibody, wherein the heavy chainnucleic acid sequence comprises SEQ ID NO: 79 and the light chainnucleic acid sequence comprises SEQ ID NO: 95, or, in each case, atleast 90%, 95%, 97%, 98%, or 99% identical to a respective SEQ ID NO,and is most preferably an IgG1, IgG2, or IgG4 isotype.

In one embodiment, the host cell is Chinese hamster ovary (CHO) cell. Inone embodiment, the host cell is a mammalian cell (e.g., a human cellsuch as an HEK293 cell, a hamster cell such as a CHO cell, etc.), abacterial cell (e.g., an E. coli cell) a yeast cell (e.g., a Pichiapastoris cell, etc.), a plant cell (e.g., a Nicotiana benthamiana cell),etc. Mammalian cells are preferred due to glycosylation patterns thatare most favorable.

The invention also provides pharmaceutical compositions comprising anantibody or antigen binding fragment of the invention and apharmaceutically acceptable carrier or diluent.

In one embodiment, the composition comprises one or more furthertherapeutic agents. In one embodiment, the further therapeutic agent isselected from the group consisting of: an anti-CD27 antibody or anantigen binding fragment thereof; an anti-LAG3 antibody or an antigenbinding fragment thereof; an anti-APRIL antibody or an antigen bindingfragment thereof; an anti-TIGIT antibody or antigen biding fragmentthereof; an anti-VISTA antibody or an antigen binding fragment thereof;an anti-BTLA antibody or an antigen binding fragment thereof; ananti-TIM3 antibody or an antigen binding fragment thereof; an anti-CTLA4antibody or an antigen binding fragment thereof; an anti-HVEM antibodyor an antigen binding fragment thereof; an anti-CD70 antibody or anantigen binding fragment thereof; an anti-CD137 antibody or an antigenbinding fragment thereof; an anti-OX40 antibody or an antigen bindingfragment thereof; an anti-CD28 antibody or an antigen binding fragmentthereof; thereof; an anti-PD1 antibody or an antigen binding fragmentthereof; an anti-PDL1 antibody or an antigen binding fragment thereof;an anti-PDL2 antibody or an antigen binding fragment thereof; ananti-GITR antibody or an antigen binding fragment thereof; an anti-ICOSantibody or an antigen binding fragment thereof; an anti-ILT2 antibodyor antigen binding fragment thereof; an anti-ILT3 antibody or antigenbinding fragment thereof; an anti-ILT4 antibody or antigen bindingfragment thereof; and an anti-ILT5 antibody or an antigen bindingfragment thereof; an anti 4-1BB antibody or an antigen binding fragmentthereof; an anti-NKG2A antibody or an antigen binding fragment thereof;an anti-NKG2C antibody or an antigen binding fragment thereof; ananti-NKG2E antibody or an antigen binding fragment thereof; an anti-TSLPantibody or an antigen binding fragment thereof; an anti-IL-10 antibodyor an antigen binding fragment thereof; IL-10 or PEGylated IL-10; anagonist (e.g., an agonistic antibody or antigen-binding fragmentthereof, or a soluble fusion) of a TNF receptor protein; anImmunoglobulin-like protein; a cytokine receptor; an integrin; asignaling lymphocytic activation molecules (SLAM proteins); anactivating NK cell receptor; a Toll like receptor; OX40; CD2; CD7; CD27;CD28; CD30; CD40; ICAM-1; LFA-1 (CD1 la/CD18); 4-1BB (CD137); B7-H3;ICOS (CD278); GITR; BAFFR; LIGHT; HVEM (LIGHTR); KIRDS2; SLAMF7; NKp80(KLRF1); NKp44; NKp30; NKp46; CD19; CD4; CD8alpha; CD8beta; IL2R beta;IL2R gamma; IL7R alpha; ITGA4; VLA1; CD49a; ITGA4; IA4; CD49D; ITGA6;VLA-6; CD49f; ITGAD; CD1 1d; ITGAE; CD103; ITGAL; ITGAM; CD1 lb; ITGAX;CD1 1c; ITGB1; CD29; ITGB2; CD18; ITGB7; NKG2D; NKG2C; TNFR2;TRANCE/RANKL; DNAM1 (CD226); SLAMF4 (CD244; 2B4); CD84; CD96 (Tactile);CEACAM1; CRTAM; Ly9 (CD229); CD160 (BY55); PSGL1; CD100 (SEMA4D); CD69;SLAMF6 (NTB-A; Ly108); SLAM (SLAMF1, CD150, IPO-3); SLAM7; BLAME(SLAMF8); SELPLG (CD162); LTBR; LAT; GADS; PAG/Cbp; CD19a; a ligand thatspecifically binds with CD83; an inhibitor of CD47, PD-1, PD-L1; PD-L2;CTLA4; TIM3; LAG3; CEACAM (e.g.; CEACAM-1, -3 and/or -5); VISTA; BTLA;TIGIT; LAIR1; IDO; TDO; CD160; TGFR beta; and a cyclic dinculeotide orother STING pathway agonist.

The invention also comprises a combination comprising an antibody orantigen binding fragment of the invention and a second antibody thatinduces ADCC, wherein said antibody or antigen binding fragment of theinvention enhances the antibody-mediated destruction of cells by thesecond antibody. Antibody-dependent cell-mediated cytotoxicity (ADCC) isa mechanism of cell-mediated immune defense whereby an effector cell ofthe immune system actively lyses a target cell, whose membrane-surfaceantigens have been bound by specific antibodies. ADCC is often thoughtof as being mediated by natural killer (NK) cells, but dendritic cells,macrophages, monocytes, and granulocytes can also mediate ADCC.

The invention also comprises a combination comprising an antibody orantigen binding fragment of the invention and a second antibody thatinduces ADCP, wherein said antibody or antigen binding fragment of theinvention enhances the antibody-mediated phagocytosis of cells by thesecond antibody. Antibody-dependent cell-mediated phagocytosis (ADCP) isa mechanism of cell-mediated immune defense whereby target cells arekilled via granulocyte, monocyte, dendritic cell, or macrophage-mediatedphagocytosis.

Natural killer (NK) cells play a major role in cancer immunotherapiesthat involve tumor-antigen targeting by monoclonal antibodies (mAbs). Inthe context of targeting cells, NK cells can be “specifically activated”through certain Fc receptors that are expressed on their cell surface.NK cells can express FcγRIIIA and/or FcγRIIC, which can bind to the Fcportion of immunoglobulins, transmitting activating signals within NKcells. Once activated through Fc receptors by antibodies bound to targetcells, NK cells are able to lyse target cells without priming, andsecrete cytokines like interferon gamma to recruit adaptive immunecells. Likewise, tumor-associated macrophages (TAMs) express surfacereceptors that bind the Fc fragment of antibodies and enable them toengage in Ab-dependent cellular cytotoxicity/phagocytosis (ADCC/ADCP).Because SIRPα/CD47 signalling induces a “don't eat me” response thatreduces ADCC/ADCP, blocking of this signaling by the anti-SIRPαantibodies or antigen binding fragments of the invention can enhanceADCC of tumor cells bearing the antigenic determinant to which thetherapeutic antibody is directed.

This ADCC/ADCP as a mode of action may be utilized in the treatment ofvarious cancers and infectious diseases. An exemplary list ofADCC/ADCP-inducing antibodies and antibody conjugates that can becombined with the antibodies or antigen binding fragments of the presentinvention includes, but is not limited to, Rituximab, ublituximab,margetuximab, IMGN-529, SCT400, veltuzumab, Obinutuzumab, ADCT-502,Hu14.18K322A, Hu3F8, Dinituximab, Trastuzumab, Cetuximab, Rituximab-RLI,c.60C3-RLI, Hu14.18-IL2, KM2812, AFM13, and (CD20)₂xCD16, erlotinib(Tarceva), daratumumab, alemtuzumab, pertuzumab, brentuximab,elotuzumab, ibritumomab, ifabotuzumab, farletuzumab, otlertuzumab,carotuximab, epratuzumab, inebilizumab, lumretuzumab, 4G7SDIE, AFM21,AFM22, LY-3022855, SNDX-6352, AFM-13, BI-836826, BMS-986012, BVX-20,mogamulizumab, ChiLob-7/4, leukotuximab, isatuximab, DS-8895, FPA144,GM102, GSK-2857916, IGN523, IT1208, ADC-1013, CAN-04, XOMA-213,PankoMab-GEX, chKM-4927, IGN003, IGN004, IGN005, MDX-1097, MOR202,MOR-208, oportuzumab, ensituximab, vedotin (Adcetris), ibritumomabtiuxetan, ABBV-838, HuMax-AXL-ADC, and ado-trastuzumab emtansine(Kadcyla). An exemplary list of target antigens for suchADCC/ADCP-inducing antibodies includes, but is not limited to, AMHR2,AXL, BCMA, CA IX, CD4, CD16, CD19, CD20, CD22, CD30, CD37, CD38, CD40,CD52, CD98, CSF1R, GD2, CCR4, CS1, EpCam, EGFR, EGFRvIII, Endoglin,EPHA2, EphA3, FGFR2b, folate receptor alpha, fucosyl-GM1, HER2, HERS,IL1RAP, kappa myeloma antigen, MS4A1, prolactin receptor, TA-MUC1, andPSMA.

In certain embodiments, the second antibody or antigen binding fragmentthereof induces ADCP. By way of example only, such antibodies may beselected from the group consisting of Rituximab, ublituximab,margetuximab, IMGN-529, SCT400, veltuzumab, Obinutuzumab, Trastuzumab,Cetuximab, alemtuzumab, ibritumomab, farletuzumab, inebilizumab,lumretuzumab, 4G7SDIE, BMS-986012, BVX-20, mogamulizumab, ChiLob-7/4,GM102, GSK-2857916, PankoMab-GEX, chKM-4927, MDX-1097, MOR202, andMOR-208.

In embodiments where the antibodies or antigen binding fragments of thepresent invention are combined with one or more ADCC/ADCP-inducingantibodies and antibody conjugates, such combinations may also be usedoptionally in association with a further therapeutic agent ortherapeutic procedure. In one embodiment, the further therapeutic agentis selected from the group consisting of: an anti-LAG3 antibody or anantigen binding fragment thereof; an anti-APRIL antibody or an antigenbinding fragment thereof; an anti-TIGIT antibody or an antigen bindingfragment thereof; an anti-VISTA antibody or an antigen binding fragmentthereof; an anti-BTLA antibody or an antigen binding fragment thereof;an anti-TIM3 antibody or an antigen binding fragment thereof; ananti-CTLA4 antibody or an antigen binding fragment thereof; an anti-HVEMantibody or an antigen binding fragment thereof; an anti-CD70 antibodyor an antigen binding fragment thereof; an anti-CD137 antibody or anantigen binding fragment thereof; an anti-OX40 antibody or an antigenbinding fragment thereof; an anti-CD28 antibody or an antigen bindingfragment thereof; thereof; an anti-PD1 antibody or an antigen bindingfragment thereof; an anti-PDL1 antibody or an antigen binding fragmentthereof; an anti-PDL2 antibody or an antigen binding fragment thereof;an anti-GITR antibody or an antigen binding fragment thereof; ananti-ICOS antibody or an antigen binding fragment thereof; an anti-ILT2antibody or antigen binding fragment thereof; an anti-ILT3 antibody orantigen binding fragment thereof; an anti-ILT4 antibody or antigenbinding fragment thereof; an anti-ILT5 antibody or an antigen bindingfragment thereof; and an anti-4-1BB antibody or an antigen bindingfragment thereof; an anti-NKG2A antibody or an antigen binding fragmentthereof; an anti-NKG2C antibody or an antigen binding fragment thereof;an anti-NKG2E antibody or an antgien binding fragment thereof; ananti-TSLP antibody or an antigen binding fragment thereof; an anti-IL-10antibody or an antigen binding fragment thereof; and IL-10 or PEGylatedIL-10.

The invention also provides a vessel or injection device comprisinganyone of the anti-SIRPα antibodies or antigen binding fragments of theinvention.

The invention also provides a method of producing an anti-SIRPα antibodyor antigen binding fragment of the invention comprising: culturing ahost cell comprising a polynucleotide encoding a heavy chain and/orlight chain of an antibody of the invention (or an antigen bindingfragment thereof) under conditions favorable to expression of thepolynucleotide; and optionally, recovering the antibody or antigenbinding fragment from the host cell and/or culture medium. In oneembodiment, the polynucleotide encoding the heavy chain and thepolynucleotide encoding the light chain are in a single vector. Inanother embodiment, the polynucleotide encoding the heavy chain and thepolynucleotide encoding the light chain are in different vectors.

The invention also provides a method of treating cancer in a subject inneed thereof, comprising administering to the subject an effectiveamount of an anti-SIRPα antibody or antigen binding fragment of theinvention, optionally in association with a further therapeutic agent ortherapeutic procedure.

In one embodiment, the subject to be treated is a human subject. In oneembodiment, the further therapeutic agent is selected from the groupconsisting of: an anti-LAG3 antibody or an antigen binding fragmentthereof; an anti-APRIL antibody or an antigen binding fragment thereof;an an anti-TIGIT antibody or an antigen binding fragment thereof; ananti-VISTA antibody or an antigen binding fragment thereof; an anti-BTLAantibody or an antigen binding fragment thereof; an anti-TIM3 antibodyor an antigen binding fragment thereof; an anti-CTLA4 antibody or anantigen binding fragment thereof; an anti-HVEM antibody or an antigenbinding fragment thereof; an anti-CD70 antibody or an antigen bindingfragment thereof; an anti-CD137 antibody or an antigen binding fragmentthereof; an anti-OX40 antibody or an antigen binding fragment thereof;an anti-CD28 antibody or an antigen binding fragment thereof; thereof;an anti-PD1 antibody or an antigen binding fragment thereof; ananti-PDL1 antibody or an antigen binding fragment thereof; an anti-PDL2antibody or an antigen binding fragment thereof; an anti-GITR antibodyor an antigen binding fragment thereof; an anti-ICOS antibody or anantigen binding fragment thereof; an anti-ILT2 antibody or antigenbinding fragment thereof; an anti-ILT3 antibody or antigen bindingfragment thereof; an anti-ILT4 antibody or antigen binding fragmentthereof; an anti-ILT5 antibody or an antigen binding fragment thereof;and an anti-4-1BB antibody or an antigen binding fragment thereof; ananti-NKG2A antibody or an antigen binding fragment thereof; ananti-NKG2C antibody or an antigen binding fragment thereof; ananti-NKG2E antibody or an antgien binding fragment thereof; an anti-TSLPantibody or an antigen binding fragment thereof; an anti-IL-10 antibodyor an antigen binding fragment thereof; and IL-10 or PEGylated IL-10.

The invention also provides a method of treating an infection orinfectious disease in a subject, comprising administering to the subjectan effective amount of an antibody or antigen binding fragment of theinvention, optionally in association with a further therapeutic agent ortherapeutic procedure. In one embodiment, the subject to be treated is ahuman subject.

In one embodiment, the further therapeutic agent is selected from thegroup consisting of: an anti-LAG3 antibody or an antigen bindingfragment thereof; an anti-APRIL antibody or an antigen binding fragmentthereof; an an anti-TIGIT antibody or an antigen binding fragmentthereof; an anti-VISTA antibody or an antigen binding fragment thereof;an anti-B TLA antibody or an antigen binding fragment thereof; ananti-TIM3 antibody or an antigen binding fragment thereof; an anti-CTLA4antibody or an antigen binding fragment thereof; an anti-HVEM antibodyor an antigen binding fragment thereof; an anti-CD70 antibody or anantigen binding fragment thereof; an anti-CD137 antibody or an antigenbinding fragment thereof; an anti-OX40 antibody or an antigen bindingfragment thereof; an anti-CD28 antibody or an antigen binding fragmentthereof; thereof; an anti-PD1 antibody or an antigen binding fragmentthereof; an anti-PDL1 antibody or an antigen binding fragment thereof;an anti-PDL2 antibody or an antigen binding fragment thereof; ananti-GITR antibody or an antigen binding fragment thereof; an anti-ICOSantibody or an antigen binding fragment thereof; an anti-ILT2 antibodyor antigen binding fragment thereof; an anti-ILT3 antibody or antigenbinding fragment thereof; an anti-ILT4 antibody or antigen bindingfragment thereof; an anti-ILT5 antibody or an antigen binding fragmentthereof; and an anti-4-1BB antibody or an antigen binding fragmentthereof; an anti-NKG2A antibody or an antigen binding fragment thereof;an anti-NKG2C antibody or an antigen binding fragment thereof; ananti-NKG2E antibody or an antien binding fragment thereof; an anti-TSLPantibody or an antigen binding fragment thereof; an anti-IL-10 antibodyor an antigen binding fragment thereof; and IL-10 or PEGylated IL-10.

The invention also provides a method for detecting the presence of aSIRPα peptide or a fragment thereof in a sample comprising contactingthe sample with an antibody or antigen binding fragment thereof of theinvention and detecting the presence of a complex between the antibodyor fragment and the peptide; wherein detection of the complex indicatesthe presence of the SIRPα peptide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts cross-reactivity of commercially available anti-hSIRPαantibodies with hSIRPβ1 and allele-specific binding to hSIRPαV1 andhSIRPαV2.

FIG. 2 depicts reactivity of KWAR23 antibody with hSIRPαV1, hSIRPαV2,hSIRPβ1, and hSIRPγ.

FIG. 3 depicts reactivity of antibody clone hSIRPα.50A for varioushSIRPα alleles.

FIG. 4 depicts the ability of hSIRPα.50A antibody to block recombinanthCD47/Fc-protein binding to cell surface expressed hSIRPα.

FIG. 5A depicts binding of hSIRP?.50A antibody to primary CD14+ enrichedmonocytes from a human donor.

FIG. 5B depicts binding of hSIRP?.50A antibody to primary CD14+ enrichedmonocytes from a second human donor.

FIG. 5C depicts the ability of hSIRP?.50A antibody to block hCD47binding to to primary CD14+ enriched monocytes from a human donor.

FIG. 5D depicts the ability of hSIRP?.50A antibody to block hCD47binding to to primary CD14+ enriched monocytes from a second humandonor.

FIG. 6A depicts binding of hSIRPα.50A antibody to primary humangranulocytes.

FIG. 6B depicts phagocytosis of tumor cells by primary humangranulocytes in the presence of rituximab plus or minus the hSIRPα.50Aantibody.

FIG. 6C depicts phagocytosis of tumor cells by primary humangranulocytes in the presence of daratumumab plus or minus the hSIRPα.50Aantibody.

FIG. 6D depicts phagocytosis of tumor cells by primary humangranulocytes in the presence of alemtuzumab plus or minus the hSIRPα.50Aantibody.

FIG. 6E depicts phagocytosis of tumor cells by primary humangranulocytes in the presence of cetuximab plus or minus the hSIRPα.50Aantibody.

FIG. 7 depicts phagocytosis of tumor cells by human macrophages in thepresence of the indicated antibody (rituximab or daratumumab) plus orminus the hSIRPα.50A antibody.

FIG. 8 depicts blocking of the hSIRPα/hCD47 interaction bymousehSIRPα.50A and humanized hSIRPα.50A antibodies to hSIRPα.

FIG. 9 depicts depicts hSIRPα.50A antibody binding to hSIRPαV1,hSIRPαV2, hSIRPβ1, hSIRPα-VβC1αC2α, hSIRPα-VaC1βC2α, andhSIRPα-VaC1αC2β.

FIG. 10A depicts an alignment of the hSIRPα and hSIRPβ1 IgV domain aminoacid sequences (SEQ ID NOS: 133-135).

FIG. 10B depicts loss of hSIRPα.50A antibody binding to hSIRPαV1(P74A).

FIG. 11 depicts binding of hSIRPα.40A and hSIRPα.50A antibodies tohSIRPαV1, hSIRPαV2, hSIRPβ1, hSIRPβL, and hSIRPγ.

FIG. 12 depicts binding of hSIRPα.40A and hSIRPα.50A antibodies tohSIRPαV1, hSIRPαV2, hSIRPαV3, hSIRPαV4, hSIRPαV5, hSIRPαV6, hSIRPαV8,and hSIRPαV9.

FIG. 13 depicts the ability of hSIRPα.40A and hSIRPα.50A antibodies toblock recombinant hCD47/Fc-protein binding to cell surface expressedhSIRPα.

FIG. 14A depicts binding of hSIRP?.40A antibody to primary CD14+enriched monocytes from a human donor.

FIG. 14B depicts binding of hSIRP?.40A antibody to primary CD14+enriched monocytes from a second human donor.

FIG. 14C depicts the ability of hSIRP?.40A antibody to block hCD47binding to to primary CD14+ enriched monocytes from a human donor.

FIG. 14D depicts the ability of hSIRP?.40A antibody to block hCD47binding to to primary CD14+ enriched monocytes from a second humandonor.

FIG. 15A depicts binding of hSIRPα.40A and hSIRPα.50A antibodies toprimary human granulocytes.

FIG. 15B depicts phagocytosis of Ramos cells by primary humangranulocytes in the presence of rituximab plus or minus the hSIRPα.40Aand hSIRPα.50A antibodies.

FIG. 16 depicts enhancement of rituximab-induced Raji cell phagocytosisby hSIRPα.40A and hSIRPα.50A antibodies.

FIG. 17 depicts binding of mouse hSIRPα.40A and humanized hSIRPα.40Aantibodies to hSIRPα.

FIG. 18 depicts the blockade of hCD47 binding to hSIRPα in the presenceof humanized hSIRPα.40A antibody variants.

FIG. 19 depicts binding of hSIRPα.40A and hSIRPα.50A antibodies tohSIRPαV1, hSIRPαV2, hSIRPβ1, hSIRP-VγC1βC2β, hSIRP-VβC1γC2β, andhSIRP-VβC1βC2γ.

FIG. 20 depicts loss of hSIRPα.40A and hSIRPα.50A antibody binding tohSIRPαV1(P74A).

FIG. 21 depicts the ability of chimeric hSIRPα.40A antibody variants toaffect rituximab-mediated phagocytosis.

FIG. 22 depicts the ability of humanized hSIRPα.40A antibody variants toaffect rituximab-mediated phagocytosis.

FIG. 23A depicts the ability of mouse hSIRPα.50A and chimeric hSIRPα.50AhIgG2 and hIgG4 antibody variants to affect rituximab-mediatedphagocytosis.

FIG. 23B depicts the ability of chimeric hSIRPα.50A hIgG2 and hIgG4antibody variants to affect rituximab-mediated phagocytosis.

FIG. 23C depicts the ability of chimerichSIRPα.50A hIgG2 and hIgG4antibody variants to affect daratumumab-mediated phagocytosis.

FIG. 23D depicts the ability of mouse hSIRPα.50A and chimeric hSIRPα.50AhIgG2 antibody variants to affect rituximab-mediated phagocytosis ingranulocytes.

FIG. 24A depicts the ability of mouse hSIRPα.50A and chimerichSIRPα.50A.hIgG1.N297Q, hSIRPα.50A.hIgG4.N297Q or hSIRPα.50A.hIgG2antibody variants to affect rituximab-mediated phagocytosis.

FIG. 24B depicts the ability of mouse hSIRPα.50A and chimerichSIRPα.50A.hIgG 1.N297Q, hSIRPα.50A.hIgG4.N297Q or hSIRPα.50A.hIgG2antibody variants to affect daratumumab-mediated phagocytosis.

FIG. 25 depicts the ability of chimeric hSIRPα.50A.hIgG1.N297Q,hSIRPα.50A hIgG1.L234A.L235A.P329G, and hSIRPα.50A hIgG2 or hIgG4antibody variants to affect rituximab-mediated phagocytosis.

DETAILED DESCRIPTION Abbreviations

Throughout the detailed description and examples of the invention thefollowing abbreviations will be used:

-   ADCC Antibody-dependent cellular cytotoxicity-   ADCP Antibody-dependent cellular phagocytosis-   CDC Complement-dependent cytotoxicity-   CDR Complementarity determining region in the immunoglobulin    variable regions, defined using the Kabat numbering system-   CHO Chinese hamster ovary-   EC₅₀ Concentration at which 50% of the total binding signal is    observed-   ELISA Enzyme-linked immunosorbant assay-   FR Antibody framework region: the immunoglobulin variable regions    excluding the CDR regions.-   HRP Horseradish peroxidase-   IFN interferon-   IC50 concentration resulting in 50% inhibition-   IgG Immunoglobulin G-   Kabat An immunoglobulin alignment and numbering system pioneered by    Elvin A. Kabat ((1991) Sequences of Proteins of Immunological    Interest, 5th Ed. Public Health Service, National Institutes of    Health, Bethesda, Md.)-   mAb or Mab or MAb Monoclonal antibody-   SEB Staphylococcus Enterotoxin B-   TT Tetanus toxoid-   V region The segment of Ig chains which is variable in sequence    between different antibodies. It extends to Kabat residue 109 in the    light chain and 113 in the heavy chain.-   VH Immunoglobulin heavy chain variable region-   VK Immunoglobulin kappa light chain variable region-   VL Immunoglobulin light chain variable region

Definitions

So that the invention may be more readily understood, certain technicaland scientific terms are specifically defined below. Unless specificallydefined elsewhere in this document, all other technical and scientificterms used herein have the meaning commonly understood by one ofordinary skill in the art to which this invention belongs.

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise.

“Administration” and “treatment,” as it applies to an animal, human,experimental subject, cell, tissue, organ, or biological fluid, refersto contact of an exogenous pharmaceutical, therapeutic, diagnosticagent, or composition to the animal, human, subject, cell, tissue,organ, or biological fluid. Treatment of a cell encompasses contact of areagent to the cell, as well as contact of a reagent to a fluid, wherethe fluid is in contact with the cell. “Administration” and “treatment”also means in vitro and ex vivo treatments, e.g., of a cell, by areagent, diagnostic, binding compound, or by another cell.

“Treat” or “treating” means to administer a therapeutic agent, such as acomposition containing any of the antibodies or antigen-bindingfragments of the present invention, internally or externally to asubject or patient having one or more disease symptoms, or beingsuspected of having a disease, for which the agent has therapeuticactivity. Typically, the agent is administered in an amount effective toalleviate one or more disease symptoms in the treated subject orpopulation, whether by inducing the regression of or inhibiting theprogression of such symptom(s) by any clinically measurable degree. Theamount of a therapeutic agent that is effective to alleviate anyparticular disease symptom may vary according to factors such as thedisease state, age, and weight of the patient, and the ability of thedrug to elicit a desired response in the subject. Whether a diseasesymptom has been alleviated can be assessed by any clinical measurementtypically used by physicians or other skilled healthcare providers toassess the severity or progression status of that symptom.

“Recombinant expression” of a protein means the transcription andtranslation of an exogenous gene in a host organism to generate theprotein, which is referred to herein as a “recombinant protein.”

SIRPα and Associated Proteins

SIRPα belongs to a class of membrane proteins known as “pairedreceptors” that contain several genes coding for proteins (e.g., SIRPα,SIRPβ1, and SIRPγ) with similar extracellular regions but differenttransmembrane and/or cytoplasmic regions having opposite (activating orinhibitory) signaling abilities. Like SIRPα, there are several examplesof paired receptors on NK cells and some on myeloid cells, including theSIRP and CD200 receptor families (Hatherley et al., Mol Cell. 2008; 31:266-277).

SIRPα contains an extracellular region that can be subdivided into threeseparate domains: the Ig-like (immunoglobulin-like) V-type (IgV),Ig-like C1-type (IgC1), and Ig-like C2-type (IgC2) domain. The IgVdomain is also known as the ligand-binding N-terminal domain of SIRPα.Like SIRPα, also the related proteins SIRPβ1 and SIRPγ comprise anextracellular region that can be subdivided into an IgV, IgC1, and IgC2domain. However, SIRPα, SIRPβ1 and SIRPγ have different cytoplasmicregions. SIRPβ1 has a very short cytoplasmic region of only 6 aminoacids and lacks signalling motifs for association with phosphatases.Instead, this protein associates with DNAX activation protein 12(DAP12), a dimeric adaptor protein that binds an amino acid with a basicside chain in the transmembrane region of SIRPβ1 and is able to transmitactivating signals through its immunoreceptor tyrosine-based activationmotif (ITAM). SIRPγ also has a short cytoplasmic region of 4 aminoacids, but it lacks a charged amino-acid side chain in the transmembraneregion and therefore does not associate with DAP12. Hence, SIRPγ isannotated as a non-signalling protein (Barclay, A. N. and Brown, M. H.,Nat Rev Immunol. 2006; 6: 457-464).

The major ligand of SIRPγ is CD47, which consists of one extracellularIgV domain, a five times transmembrane-spanning domain, and a shortcytoplasmic tail. CD47 functions as a cellular ligand with bindingmediated through the NH2-terminal IgV domain of SIRPγ. Evidence thatCD47 contributes to recognition of self comes from the observation thatsplenic macrophages derived from CD47-expressing mice clear infusedblood cells from CD47^(−/−) mice (Oldenborg et al., Science. 2000; 288:2051-2054).

In addition to CD47, two other SIRPγ ligands have been reported, knownas surfactant proteins A and D (Sp-A and Sp-D), both of which belong tothe collectin family. Sp-D has been reported to bind to themembrane-proximal IgC2 domain of SIRPγ in a calcium- andsaccharide-dependent manner. It is thought that Sp-A and Sp-D helpmaintain an anti-inflammatory environment in the lung by stimulatingSIRPγ on alveolar macrophages (Gardai et al., Cell. 2003; 115: 13-23).

The amino acid sequence of eight human SIRPγ variants are listed in SEQID NOs: 34, 36, 44, 46, 48, 50, 52, and 54; exemplary nucleic acidsequences encoding these variants are listed in SEQ ID NOs: 33, 35, 43,45, 47, 49, 51, and 53, respectively.

For comparison, the amino acid sequence of human SIRPβ1 and SIRPγ arelisted in SEQ ID NOs: 38 and 40, respectively, and exemplary nucleicacid sequences in SEQ ID NOs: 37 and 39, respectively.

The amino acid sequence of human CD47 is listed in SEQ ID NO: 42, and anexemplary nucleic acid sequence in SEQ ID NO: 41.

Modified SIRPγ polypeptides hSIRPα-VβC1αC2α, hSIRPα-VaC1βC2α,hSIRPα-VαC1αC2β, and hSIRPαV1(P74A) discussed hereinafter are listed inSEQ IDNOs: 56, 58, 60, and 62; exemplary nucleic acid sequences encodingthese variants are listed in SEQ ID NOs: 55, 57, 59, and 61,respectively.

Anti-SIRPα Antibodies and Antigen-Binding Fragments Thereof

The present invention provides antibodies or antigen-binding fragmentsthereof that bind human SIRPα and uses of such antibodies or fragments.In some embodiments, the anti-SIRPα antibodies are isolated.

Whether an antibody specifically binds to a polypeptide sequence (e.g.,human SIRPα, hSIRPβ1, etc.) can be determined using any assay known inthe art. Examples of assays known in the art to determining bindingaffinity include surface plasmon resonance (e.g., BIACORE) or a similartechnique (e.g. KinExa or OCTET).

As used herein, the term “antibody” refers to any form of antibody thatexhibits the desired biological activity. The term antibody includesantigen-binding portions, i.e., “antigen binding sites,” (e.g.,fragments, subsequences, complementarity determining regions (CDRs))that retain capacity to bind antigen, including (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H)1domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H)1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Single chain antibodies are also included byreference in the term “antibody.” Preferred therapeutic antibodies areintact IgG antibodies. The term “intact IgG” as used herein is meant asa polypeptide belonging to the class of antibodies that aresubstantially encoded by a recognized immunoglobulin gamma gene. Inhumans this class comprises IgG1, IgG2, IgG3, and IgG4. In mice thisclass comprises IgG1, IgG2a, IgG2b, IgG3. The known Ig domains in theIgG class of antibodies are V_(H), Cγ1, Cγ2, Cγ3, V_(L), and C_(L).

The present invention includes anti-SIRPα antigen-binding fragments andmethods of use thereof.

As used herein, a “full length antibody” is, in the case of an IgG, abivalent molecule comprising two heavy chains and two light chains. Eachheavy chain comprises a V_(H) domain followed by a constant domain(C_(H1)), a hinge region, and two more constant (C_(H2) and C_(H3))domains; while each light chain comprises one V_(L) domain and oneconstant (C_(L)) domain. A full length antibody in the case of an IgM isa decavalent or dodecavalent molecule comprising 5 or 6 linkedimmunoglobulins in which immunoglobulin each monomer has two antigenbinding sites formed of a heavy and light chain.

As used herein, unless otherwise indicated, “antibody fragment” or“antigen-binding fragment” refers to antigen-binding fragments ofantibodies, i.e. antibody fragments that retain the ability to bindspecifically to the antigen bound by the full-length antibody, e.g.fragments that retain one or more CDR regions. Examples ofantigen-binding fragments include, but are not limited to, Fab, Fab′,F(ab′)₂, and Fv fragments; diabodies; linear antibodies; single-chainantibody molecules, e.g., sc-Fv; nanobodies and multispecific antibodiesformed from antibody fragments.

The present invention includes anti-SIRPα Fab fragments and methods ofuse thereof. A “Fab fragment” is comprised of one light chain and theC_(H)1 and variable regions of one heavy chain. The heavy chain of a Fabmolecule cannot form a disulfide bond with another heavy chain molecule.A “Fab fragment” can be the product of papain cleavage of an antibody.

The present invention includes anti-SIRPα antibodies and antigen-bindingfragments thereof which comprise an Fc region and methods of usethereof. An “Fc” region contains two heavy chain fragments comprisingthe C_(H)3 and C_(H)2 domains of an antibody. The two heavy chainfragments are held together by two or more disulfide bonds and byhydrophobic interactions of the C_(H)3 domains.

The present invention includes anti-SIRPα Fab′ fragments and methods ofuse thereof. A “Fab′ fragment” contains one light chain and a portion orfragment of one heavy chain that contains the VH domain and the C_(H)1domain and also the region between the C_(H)1 and C_(H)2 domains, suchthat an interchain disulfide bond can be formed between the two heavychains of two Fab′ fragments to form a F(ab′)₂ molecule.

The present invention includes anti-SIRPα F(ab′)₂ fragments and methodsof use thereof. A “F(ab′)₂ fragment” contains two light chains and twoheavy chains containing a portion of the constant region between theC_(H1) and C_(H2) domains, such that an interchain disulfide bond isformed between the two heavy chains. A F(ab′)₂ fragment thus is composedof two Fab′ fragments that are held together by a disulfide bond betweenthe two heavy chains. An “F(ab′)₂ fragment” can be the product of pepsincleavage of an antibody.

The present invention includes anti-SIRPα Fv fragments and methods ofuse thereof. The “Fv region” comprises the variable regions from boththe heavy and light chains, but lacks the constant regions.

The present invention includes anti-SIRPα scFv fragments and methods ofuse thereof. The term “single-chain Fv” or “scFv” antibody refers toantibody fragments comprising the V_(H) and V_(L) domains of anantibody, wherein these domains are present in a single polypeptidechain. Generally, the Fv polypeptide further comprises a polypeptidelinker between the V_(H) and V_(L) domains which enables the scFv toform the desired structure for antigen-binding. For a review of scFv,see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.See also, International Patent Application Publication No. WO 88/01649and U.S. Pat. Nos. 4,946, 778 and 5,260,203.

The present invention includes anti-SIRPα domain antibodies and methodsof use thereof. A “domain antibody” is an immunologically functionalimmunoglobulin fragment containing only the variable region of a heavychain or the variable region of a light chain. In some instances, two ormore V_(H) regions are covalently joined with a peptide linker to createa bivalent domain antibody. The two V_(H) regions of a bivalent domainantibody may target the same or different antigens.

The present invention includes anti-SIRPα bivalent antibodies andmethods of use thereof. A “bivalent antibody” comprises twoantigen-binding sites. In some instances, the two binding sites have thesame antigen specificities. However, bivalent antibodies may bebispecific (see below).

The present invention includes anti-SIRPα diabodies and methods of usethereof. As used herein, the term “diabodies” refers to small antibodyfragments with two antigen-binding sites, which fragments comprise aheavy chain variable domain (V_(H)) connected to a light chain variabledomain (V_(L)) in the same polypeptide chain (V_(H)-V_(L) orV_(L)-V_(H)). By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described more fully in, e.g., EP404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci.USA 90: 6444-6448. Duobodies are described in Labrijn et al., 2013,Proc. Natl. Acad. Sci. USA 110 (13): 5145-5150. For a review ofengineered antibody variants generally see Holliger and Hudson (2005)Nat. Biotechnol. 23:1126-1136.

Typically, an antibody or antigen-binding fragment of the inventionwhich is modified in some way retains at least 10% of its bindingactivity (when compared to the parental antibody) when that activity isexpressed on a molar basis. Preferably, an antibody or antigen-bindingfragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95%or 100% or more of the SIRPα binding affinity as the parental antibody.It is also intended that an antibody or antigen-binding fragment of theinvention can include conservative or non-conservative amino acidsubstitutions (referred to as “conservative variants” or “functionconserved variants” of the antibody) that do not substantially alter itsbiologic activity.

The present invention includes isolated anti-SIRPα antibodies andantigen-binding fragments thereof and methods of use thereof. Herein,the term “isolated” is not intended to refer to a complete absence ofsuch biological molecules or to an absence of water, buffers, or saltsor to components of a pharmaceutical formulation that includes theantibodies or fragments. An “isolated” antibody, antigen-bindingfragment, nucleic acid, etc., is one which has been identified andseparated and/or recovered from one or more components of its naturalenvironment. In preferred embodiments, the antibody, antigen-bindingfragment, nucleic acid, etc., is purified to 75% by weight or more, morepreferably to 90% by weight or more, still more preferably to 95% byweight or more, an still more preferably to 98% by weight or more. Thus,“isolated” biological molecules are at least partially free of otherbiological molecules from the cells or cell cultures in which they areproduced. Such biological molecules include nucleic acids, proteins,lipids, carbohydrates, or other material such as cellular debris andgrowth medium. An isolated antibody or antigen-binding fragment mayfurther be at least partially free of expression system components suchas biological molecules from a host cell or of the growth mediumthereof.

The present invention includes anti-SIRPα chimeric antibodies (e.g.,human constant domain/mouse variable domain) and methods of use thereof.As used herein, a “chimeric antibody” is an antibody having the variabledomain from a first antibody and the constant domain from a secondantibody, where the first and second antibodies are from differentspecies. (U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc.Natl. Acad. Sci. USA 81: 6851-6855). Typically, the variable domains areobtained from an antibody from an experimental animal (the “parentalantibody”), such as a rodent, and the constant domain sequences areobtained from human antibodies, so that the resulting chimeric antibodywill be less likely to elicit an adverse immune response in a humansubject than the parental (e.g., mouse) antibody.

The present invention includes anti-SIRPα humanized antibodies andantigen-binding fragments thereof (e.g., rat or mouse antibodies thathave been humanized) and methods of use thereof. As used herein, theterm “humanized antibody” refers to forms of antibodies that containsequences from both human and non-human (e.g., mouse or rat) antibodies.In general, the humanized antibody will comprise substantially of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin, and all or substantially all of the framework(FR) regions are those of a human immunoglobulin sequence. The humanizedantibody may optionally comprise at least a portion of a humanimmunoglobulin constant region (Fc). For more details about humanizedantibodies, see, e.g., Jones et al., Nature, 321:522-525 (1986);Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op. Struct.Biol., 2:593-596 (1992); and Clark, Immunol. Today 21: 397-402 (2000).

In general, the basic antibody structural unit comprises a tetramer.Each tetramer includes two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of the heavy chain maydefine a constant region primarily responsible for effector function.Typically, human light chains are classified as kappa and lambda lightchains. Furthermore, human heavy chains are typically classified as mu,delta, gamma, alpha, or epsilon, and define the antibody's isotype asIgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavychains, the variable and constant regions are joined by a “J” region ofabout 12 or more amino acids, with the heavy chain also including a “D”region of about 10 more amino acids. See generally, FundamentalImmunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, in general, an intact antibody has two bindingsites. Except in bifunctional or bispecific antibodies, the two bindingsites are, in general, the same.

Typically, the variable domains of both the heavy and light chainscomprise three hypervariable regions, also called complementaritydetermining regions (CDRs), located within relatively conservedframework regions (FR). The CDRs are usually aligned by the frameworkregions, enabling binding to a specific epitope. In general, fromN-terminal to C-terminal, both light and heavy chains variable domainscomprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment ofamino acids to each domain is, generally, in accordance with thedefinitions of Sequences of Proteins of Immunological Interest, Kabat,et al.; National Institutes of Health, Bethesda, Md.; 5^(th) ed.; NIHPubl. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat,et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) JMol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody or antigen-binding fragment thereof that areresponsible for antigen-binding. The hypervariable region comprisesamino acid residues from a “complementarity determining region” or “CDR”(i.e. CDRL1, CDRL2 and CDRL3 in the light chain variable domain andCDRH1, CDRH2 and CDRH3 in the heavy chain variable domain). See Kabat etal. (1991) Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(defining the CDR regions of an antibody by sequence); see also Chothiaand Lesk (1987) J. Mol. Biol. 196: 901-917 (defining the CDR regions ofan antibody by structure). As used herein, the term “framework” or “FR”residues refers to those variable domain residues other than thehypervariable region residues defined herein as CDR residues.

“Isolated nucleic acid molecule” or “isolated polynucleotide” means aDNA or RNA of genomic, mRNA, cDNA, or synthetic origin or somecombination thereof which is not associated with all or a portion of apolynucleotide in which the isolated polynucleotide is found in nature,or is linked to a polynucleotide to which it is not linked in nature.For purposes of this disclosure, it should be understood that “a nucleicacid molecule comprising” a particular nucleotide sequence does notencompass intact chromosomes. Isolated nucleic acid molecules“comprising” specified nucleic acid sequences may include, in additionto the specified sequences, coding sequences for up to ten or even up totwenty or more other proteins or portions or fragments thereof, or mayinclude operably linked regulatory sequences that control expression ofthe coding region of the recited nucleic acid sequences, and/or mayinclude vector sequences.

The phrase “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to use promoters,polyadenylation signals, and enhancers.

A nucleic acid or polynucleotide is “operably linked” when it is placedinto a functional relationship with another nucleic acid sequence. Forexample, DNA for a presequence or secretory leader is operably linked toDNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Generally, but not always, “operably linked” means that the DNAsequences being linked are contiguous, and, in the case of a secretoryleader, contiguous and in reading phase. However, enhancers do not haveto be contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that not all progeny willhave precisely identical DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

As used herein, “germline sequence” refers to a sequence of unrearrangedimmunoglobulin DNA sequences. Any suitable source of unrearrangedimmunoglobulin sequences may be used. Human germline sequences may beobtained, for example, from JOINSOLVER germline databases on the websitefor the National Institute of Arthritis and Musculoskeletal and SkinDiseases of the United States National Institutes of Health. Mousegermline sequences may be obtained, for example, as described inGiudicelli et al. (2005) Nucleic Acids Res. 33: D256-D261.

Physical and Functional Properties of the Exemplary Anti-SIRPαAntibodies

The present invention provides anti-SIRPα antibodies and antigen-bindingfragments thereof having specified structural and functional features,and methods of use of the antibodies or antigen-binding fragmentsthereof in the treatment or prevention of disease (e.g., cancer orinfectious disease).

As stated above, antibodies and fragments that bind to the same epitopeas any of the anti-SIRPα antibodies or antigen-binding fragments thereofof the present invention also form part of the present invention. In oneembodiment, the invention provides an antibody or antigen bindingfragment thereof that binds to the same epitope of human SIRPα as anantibody comprising one of the following combinations of heavy chainsequence/light chain sequence (or in each case an amino acid sequence atleast 90%, 95%, 97%, 98%, or 99% identical thereto):

SEQ ID NO: 10/SEQ ID NO: 20 (referred to herein as hSIRPα.50A.H1L1)

SEQ ID NO: 10/SEQ ID NO: 22 (referred to herein as hSIRPα.50A.H1L2)

SEQ ID NO: 10/SEQ ID NO: 24 (referred to herein as hSIRPα.50A.H1L3)

SEQ ID NO: 10/SEQ ID NO: 26 (referred to herein as hSIRPα.50A.H1L4)

SEQ ID NO: 10/SEQ ID NO: 28 (referred to herein as hSIRPα.50A.H1L5)

SEQ ID NO: 12/SEQ ID NO: 20 (referred to herein as hSIRPα.50A.H2L1)

SEQ ID NO: 12/SEQ ID NO: 22 (referred to herein as hSIRPα.50A.H2L2)

SEQ ID NO: 12/SEQ ID NO: 24 (referred to herein as hSIRPα.50A.H2L3)

SEQ ID NO: 12/SEQ ID NO: 26 (referred to herein as hSIRPα.50A.H2L4)

SEQ ID NO: 12/SEQ ID NO: 28 (referred to herein as hSIRPα.50A.H2L5)

SEQ ID NO: 14/SEQ ID NO: 20 (referred to herein as hSIRPα.50A.H3L1)

SEQ ID NO: 14/SEQ ID NO: 22 (referred to herein as hSIRPα.50A.H3L2)

SEQ ID NO: 14/SEQ ID NO: 24 (referred to herein as hSIRPα.50A.H3L3)

SEQ ID NO: 14/SEQ ID NO: 26 (referred to herein as hSIRPα.50A.H3L4)

SEQ ID NO: 14/SEQ ID NO: 28 (referred to herein as hSIRPα.50A.H3L5)

SEQ ID NO: 16/SEQ ID NO: 20 (referred to herein as hSIRPα.50A.H4L1)

SEQ ID NO: 16/SEQ ID NO: 22 (referred to herein as hSIRPα.50A.H4L2)

SEQ ID NO: 16/SEQ ID NO: 24 (referred to herein as hSIRPα.50A.H4L3)

SEQ ID NO: 16/SEQ ID NO: 26 (referred to herein as hSIRPα.50A.H4L4)

SEQ ID NO: 16/SEQ ID NO: 28 (referred to herein as hSIRPα.50A.H4L5)

SEQ ID NO: 18/SEQ ID NO: 20 (referred to herein as hSIRPα.50A.H5L1)

SEQ ID NO: 18/SEQ ID NO: 22 (referred to herein as hSIRPα.50A.H5L2)

SEQ ID NO: 18/SEQ ID NO: 24 (referred to herein as hSIRPα.50A.H5L3)

SEQ ID NO: 18/SEQ ID NO: 26 (referred to herein as hSIRPα.50A.H5L4)

SEQ ID NO: 18/SEQ ID NO: 28 (referred to herein as hSIRPα.50A.H5L5)

SEQ ID NO: 78/SEQ ID NO: 90 (referred to herein as hSIRPα.40A.H1L1)

SEQ ID NO: 78/SEQ ID NO: 92 (referred to herein as hSIRPα.40A.H1L2)

SEQ ID NO: 78/SEQ ID NO: 94 (referred to herein as hSIRPα.40A.H1L3)

SEQ ID NO: 78/SEQ ID NO: 96 (referred to herein as hSIRPα.40A.H1L4)

SEQ ID NO: 78/SEQ ID NO: 98 (referred to herein as hSIRPα.40A.H1L5)

SEQ ID NO: 78/SEQ ID NO: 100 (referred to herein as hSIRPα.40A.H1L6)

SEQ ID NO: 80/SEQ ID NO: 90 (referred to herein as hSIRPα.40A.H2L1)

SEQ ID NO: 80/SEQ ID NO: 92 (referred to herein as hSIRPα.40A.H2L2)

SEQ ID NO: 80/SEQ ID NO: 94 (referred to herein as hSIRPα.40A.H2L3)

SEQ ID NO: 80/SEQ ID NO: 96 (referred to herein as hSIRPα.40A.H2L4)

SEQ ID NO: 80/SEQ ID NO: 98 (referred to herein as hSIRPα.40A.H2L5)

SEQ ID NO: 80/SEQ ID NO: 100 (referred to herein as hSIRPα.40A.H2L6)

SEQ ID NO: 82/SEQ ID NO: 90 (referred to herein as hSIRPα.40A.H3L1)

SEQ ID NO: 82/SEQ ID NO: 92 (referred to herein as hSIRPα.40A.H3L2)

SEQ ID NO: 82/SEQ ID NO: 94 (referred to herein as hSIRPα.40A.H3L3)

SEQ ID NO: 82/SEQ ID NO: 96 (referred to herein as hSIRPα.40A.H3L4)

SEQ ID NO: 82/SEQ ID NO: 98 (referred to herein as hSIRPα.40A.H3L5)

SEQ ID NO: 82/SEQ ID NO: 100 (referred to herein as hSIRPα.40A.H3L6)

SEQ ID NO: 84/SEQ ID NO: 90 (referred to herein as hSIRPα.40A.H4L1)

SEQ ID NO: 84/SEQ ID NO: 92 (referred to herein as hSIRPα.40A.H4L2)

SEQ ID NO: 84/SEQ ID NO: 94 (referred to herein as hSIRPα.40A.H4L3)

SEQ ID NO: 84/SEQ ID NO: 96 (referred to herein as hSIRPα.40A.H4L4)

SEQ ID NO: 84/SEQ ID NO: 98 (referred to herein as hSIRPα.40A.H4L5)

SEQ ID NO: 84/SEQ ID NO: 100 (referred to herein as hSIRPα.40A.H4L6)

SEQ ID NO: 86/SEQ ID NO: 90 (referred to herein as hSIRPα.40A.H5L1)

SEQ ID NO: 86/SEQ ID NO: 92 (referred to herein as hSIRPα.40A.H5L2)

SEQ ID NO: 86/SEQ ID NO: 94 (referred to herein as hSIRPα.40A.H5L3)

SEQ ID NO: 86/SEQ ID NO: 96 (referred to herein as hSIRPα.40A.H5L4)

SEQ ID NO: 86/SEQ ID NO: 98 (referred to herein as hSIRPα.40A.H5L5)

SEQ ID NO: 86/SEQ ID NO: 100 (referred to herein as hSIRPα.40A.H5L6)

SEQ ID NO: 88/SEQ ID NO: 90 (referred to herein as hSIRPα.40A.H6L1)

SEQ ID NO: 88/SEQ ID NO: 92 (referred to herein as hSIRPα.40A.H6L2)

SEQ ID NO: 88/SEQ ID NO: 94 (referred to herein as hSIRPα.40A.H6L3)

SEQ ID NO: 88/SEQ ID NO: 96 (referred to herein as hSIRPα.40A.H6L4)

SEQ ID NO: 88/SEQ ID NO: 98 (referred to herein as hSIRPα.40A.H6L5)

SEQ ID NO: 88/SEQ ID NO: 100 (referred to herein as hSIRPα.40A.H6L6).

There are several methods available for mapping antibody epitopes ontarget antigens, including: H/D-Ex mass spectrometry, crosslinkingcoupled mass spectrometry, X-ray crystallography, pepscan analysis andsite directed mutagenesis. For example, HDX (Hydrogen DeuteriumExchange) coupled with proteolysis and mass spectrometry can be used todetermine the epitope of an antibody on a specific antigen Y. HDX-MSrelies on the accurate measurement and comparison of the degree ofdeuterium incorporation by an antigen when incubated in D20 on its ownand in presence of its antibody at various time intervals. Deuterium isexchanged with hydrogen on the amide backbone of the proteins in exposedareas whereas regions of the antigen bound to the antibody will beprotected and will show less or no exchange after analysis by LC-MS/MSof proteolytic fragments., Crosslinking coupled mass spectrometry beginsby binding the antibody and the antigen with a mass labeled chemicalcrosslinker. Next the presence of the complex is confirmed using highmass MALDI detection. Because after crosslinking chemistry the Ab/Agcomplex is extremely stable, many various enzymes and digestionconditions can be applied to the complex to provide many differentoverlapping peptides. Identification of these peptides is performedusing high resolution mass spectrometry and MS/MS techniques.Identification of the crosslinked peptides is determined using mass taglinked to the cross-linking reagents. After MS/MS fragmentation and dataanalysis, both epitope and paratope are determined in the sameexperiment.

The scope of the present invention also includes isolated anti-SIRPαantibodies and antigen-binding fragments thereof (e.g., humanizedantibodies), comprising a variant of an immunoglobulin chain set forthherein, wherein the variant exhibits one or more of the followingproperties:

-   -   binds human SIRPαV1 protein having the sequence of SEQ ID NO: 34        with an EC₅₀<1 nM; and exhibits at least a 100-fold higher EC₅₀        for SIRPαV1(P74A) having the sequence of SEQ ID NO: 62; and        optionally also at least a 100-fold higher EC₅₀ for human SIRPβ1        protein having the sequence of SEQ ID NO: 38 (in each case        wherein the reduced EC₅₀ is relative to the EC₅₀ for human        SIRPαV1 protein having the sequence of SEQ ID NO: 34, and in        each case preferably when measured by cellular ELISA (CELISA) as        described hereinafter;    -   binds to a cell expressing human SIRPαV1 protein with an EC₅₀<10        nM, preferably <5 nM, more preferably <1.5 nM, still more        preferably <1.0 nM, even more preferably <0.5 nM, and most        preferably about 0.3nM or less;    -   binds to a cell expressing human SIRPαV2 protein with an EC₅₀<10        nM, preferably <5 nM, more preferably <1.5 nM, still more        preferably <1.0 nM, even more preferably <0.5 nM, and most        preferably about 0.3nM or less;    -   does not appreciably bind to SIRPβ1 protein at an antibody        concentration of 50 nM, preferably 67 nM, and more preferably        100 nM; or alternatively at a concentration that is 10-fold        greater, preferably 50-fold greater, more preferably 100-fold        greater, and still more preferably 200-fold greater than the        antibody's EC₅₀ for SIRPαV1 or SIRPαV2;    -   inhibits binding between human SIRPα and CD47 with an IC₅₀<10.0        nM, more preferably <5.0 nM, still more preferably <2.5 nM, and        most preferably about 1.0 nM or less; and    -   exhibits a T20 “humanness” of at least 79, and more preferably        85%.

In other embodiments, the invention provides antibodies orantigen-binding fragment thereof that bind human SIRPα (e.g., humanizedantibodies) and have V_(H) domains and V_(L) domains with at least 90%sequence identity with SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 102, 7,10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8, 20,22, 24, 26, 28, and 32. In other embodiments, the invention providesantibodies or antigen-binding fragment thereof that bind human SIRPα(e.g., humanized antibodies) and have V_(H) domains and V_(L) domainswith at least 95% sequence identity with SEQ ID NOs: 75, 78, 80, 82, 84,86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98,100, 104, 8, 20, 22, 24, 26, 28, and 32. In other embodiments, theinvention provides antibodies or antigen-binding fragment thereof thatbind human SIRPα (e.g., humanized antibodies) and have V_(H) domains andV_(L) domains with at least 97% sequence identity with SEQ ID NOs: 75,78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and 76, 90,92, 94, 96, 98, 100, 104, 8, 20, 22, 24, 26, 28, and 32. In otherembodiments, the invention provides antibodies or antigen-bindingfragment thereof that bind human SIRPα (e.g., humanized antibodies) andhave V_(H) domains and V_(L) domains with at least 98% sequence identitywith SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18,and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8, 20, 22, 24, 26, 28, and32. In other embodiments, the invention provides antibodies orantigen-binding fragment thereof that bind human SIRPα (e.g., humanizedantibodies) and have V_(H) domains and V_(L) domains with at least 99%sequence identity with SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 102, 7,10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8, 20,22, 24, 26, 28, and 32. Preferably, in each case, the sequencedifferences between SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10,12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8, 20, 22,24, 26, 28, and 32 and the variants consist of conservativesubstitutions and are most preferably limited to substitutions withinthe framework residues.

The following references relate to BLAST algorithms often used forsequence analysis: BLAST ALGORITHMS: Camacho, C. et al. (2009): BMCBioinformatics 10:421; Altschul et al. (2005) FEBS J. 272(20):5101-5109; Altschul, S. F., et al., (1990) J. Mol. Biol. 215:403-410;Gish, W., et al., (1993) Nature Genet. 3:266-272; Madden, T. L., et al.,(1996) Meth. Enzymol. 266:131-141; Altschul, S. F., et al., (1997)Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997) Genome Res.7:649-656; Wootton, J. C., et al., (1993) Comput. Chem. 17:149-163;Hancock, J. M. et al., (1994) Comput. Appl. Biosci. 10:67-70; ALIGNMENTSCORING SYSTEMS: Dayhoff, M. O., et al., “A model of evolutionary changein proteins.” in Atlas of Protein Sequence and Structure, (1978) vol. 5,suppl. 3. M. O. Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found.,Washington, D.C.; Schwartz, R. M., et al., “Matrices for detectingdistant relationships.” in Atlas of Protein Sequence and Structure,(1978) vol. 5, suppl. 3.” M. O. Dayhoff (ed.), pp. 353-358, Natl.Biomed. Res. Found., Washington, D.C.; Altschul, S. F., (1991) J. Mol.Biol. 219:555-565; States, D. J., et al., (1991) Methods 3:66-70;Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919;Altschul, S. F., et al., (1993) J. Mol. Evol. 36:290-300; ALIGNMENTSTATISTICS: Karlin, S., et al., (1990) Proc. Natl. Acad. Sci. USA87:2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA90:5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22:2022-2039; andAltschul, S. F. “Evaluating the statistical significance of multipledistinct local alignments.” in Theoretical and Computational Methods inGenome Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, New York. Inthe present application, percent identity comparisons are preferablyperformed by a BLAST algorithm wherein the parameters of the algorithmare selected to give the largest match between the respective sequencesover the entire length of the respective reference sequences (e.g.expect threshold: 10; word size: 6; max matches in a query range: 0;BLOSUM 62 matrix; gap costs: existence 11, extension 1; conditionalcompositional score matrix adjustment).

“Conservatively modified variants” or “conservative substitution” refersto substitutions of amino acids in a protein with other amino acidshaving similar characteristics (e.g. charge, side-chain size,hydrophobicity/hydrophilicity, backbone conformation and rigidity,etc.), such that the changes can frequently be made without altering thebiological activity of the protein. Those of skill in this art recognizethat, in general, single amino acid substitutions in non-essentialregions of a polypeptide do not substantially alter biological activity(see, e.g., Watson et al. (1987) Molecular Biology of the Gene, TheBenjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition,substitutions of structurally or functionally similar amino acids areless likely to disrupt biological activity. Exemplary conservativesubstitutions are set forth the following Table 1.

TABLE 1 Exemplary Conservative Amino Acid Substitutions Original residueConservative substitution Ala (A) Gly; Ser Arg (R) Lys; His Asn (N) Gln;His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly(G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) ThrThr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

Function-conservative variants of the antibodies of the invention arealso contemplated by the present invention. “Function-conservativevariants,” as used herein, refers to antibodies or fragments in whichone or more amino acid residues have been changed without altering adesired property, such an antigen affinity and/or specificity. Suchvariants include, but are not limited to, replacement of an amino acidwith one having similar properties, such as the conservative amino acidsubstitutions of Table 1. Also provided are isolated polypeptidescomprising the V_(L) domains of the anti-SIRPα antibodies of theinvention (e.g., SEQ ID NOs: 76, 90, 92, 94, 96, 98, 100, 8, 20, 22, 24,26, 28, and 32), and isolated polypeptides comprising the V_(H) domainsof the anti-SIRPα antibodies of the invention (e.g., SEQ ID NOs: 75, 78,80, 82, 84, 86, 88, 7, 10, 12, 14, 16, 18, and 30) having up to 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, and preferablyconservative substitutions.

The present invention further comprises the polynucleotides encoding anyof the polypeptides or immunoglobulin chains of anti-SIRPα antibodiesand antigen-binding fragments thereof of the invention. For example, thepresent invention includes the polynucleotides encoding the amino acidsdescribed in any one of SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 102, 7,10, 12, 14, 16, 18, and 30; and SEQ ID NOs: 76, 90, 92, 94, 96, 98, 100,104, 8, 20, 22, 24, 26, 28, and 32.

In one embodiment, an isolated polynucleotide, for example DNA, encodingthe polypeptide chains of the isolated antibodies or antigen-bindingfragments set forth herein is provided. In one embodiment, the isolatedpolynucleotide encodes an antibody or antigen-binding fragment thereofcomprising at least one mature immunoglobulin light chain variable (VL)domain according to the invention and/or at least one matureimmunoglobulin heavy chain variable (VH) domain according to theinvention. In some embodiments, the isolated polynucleotide encodes botha light chain and a heavy chain on a single polynucleotide molecule, andin other embodiments the light and heavy chains are encoded on separatepolynucleotide molecules. In another embodiment, the polynucleotidesfurther encodes a signal sequence.

This present invention also provides vectors, e.g., expression vectors,such as plasmids, comprising the isolated polynucleotides of theinvention, wherein the polynucleotide is operably linked to controlsequences that are recognized by a host cell when the host cell istransfected with the vector. Also provided are host cells comprising avector of the present invention and methods for producing the antibodyor antigen-binding fragment thereof or polypeptide disclosed hereincomprising culturing a host cell harboring an expression vector or anucleic acid encoding the immunoglobulin chains of the antibody orantigen-binding fragment thereof in culture medium, and isolating theantigen or antigen-binding fragment thereof from the host cell orculture medium.

Binding Affinity

By way of example, and not limitation, the antibodies andantigen-binding fragments disclosed herein may bind human SIRPαbivalently with a K_(D) value of 10×10⁻⁹M or lower) as determined bysurface plasmon resonance (e.g., BIACORE) or a similar technique (e.g.KinExa or bio-layer interferometry (OCTET)). In one embodiment, theantibodies and antigen-binding fragments disclosed herein may bind humanSIRPα or bivalently with a K_(D) value of about 5-10×10⁻⁹ M asdetermined by surface plasmon resonance (e.g., BIACORE) or a similartechnique (e.g. KinExa or OCTET). Affinity is calculated asK_(D)=k_(off)/k_(on) (k_(off) is the dissociation rate constant, K_(on)is the association rate constant and K_(D) is the equilibrium constant).Affinity can be determined at equilibrium by measuring the fractionbound (r) of labeled ligand at various concentrations (c). The data aregraphed using the Scatchard equation: r/c=K(n−r): where r=moles of boundligand/mole of receptor at equilibrium; c=free ligand concentration atequilibrium; K=equilibrium association constant; and n=number of ligandbinding sites per receptor molecule. By graphical analysis, r/c isplotted on the Y-axis versus r on the X-axis, thus producing a Scatchardplot. Antibody affinity measurement by Scatchard analysis is well knownin the art. See, e.g., van Erp et al., J. Immunoassay 12: 425-43, 1991;Nelson and Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.

Humanness

For purposes of this document, “humanness” is measured using the T20score analyzer to quantify the humanness of the variable region ofmonoclonal antibodies as described in Gao S H, Huang K, Tu H, Adler A S.Monoclonal antibody humanness score and its applications. BMCBiotechnology. 2013: 13:55. doi:10.1186/1472-6750-13-55).

A web-based tool is provided to calculate the T20 score of antibodysequences using the T20 Cutoff Human Databases:http://abAnalyzer.lakepharma.com. In computing a T20 score, an input VH,VK, or VL variable region protein sequence is first assigned Kabatnumbering, and CDR residues are identified. The full-length sequence orthe framework only sequence (with CDR residues removed) is compared toevery sequence in a respective antibody database using the blastpprotein-protein BLAST algorithm. The sequence identity between eachpairwise comparison is isolated, and after every sequence in thedatabase has been analyzed, the sequences are sorted from high to lowbased on the sequence identity to the input sequence. The percentidentity of the Top 20 matched sequences is averaged to obtain the T20score.

For each chain type (VH, VK, VL) and sequence length (full-length orframework only) in the “All Human Databases,” each antibody sequence wasscored with its respective database using the T20 score analyzer. TheT20 score was obtained for the top 20 matched sequences after the inputsequence itself was excluded (the percent identity of sequences 2through 21 were averaged since sequence 1 was always the input antibodyitself). The T20 scores for each group were sorted from high to low. Thedecrease in score was roughly linear for most of the sequences; howeverthe T20 scores for the bottom ˜15% of antibodies started decreasingsharply. Therefore, the bottom 15 percent of sequences were removed andthe remaining sequences formed the T20 Cutoff Human Databases, where theT20 score cutoff indicates the lowest T20 score of a sequence in the newdatabase.

As used herein, a “Human” antibody is one that has a T20 humanness scoreof at least 79%, and more preferably at least 85%.

Ability of Anti-hSIRPα Antibodies to Block Binding to CD47

In some embodiments, the anti-SIRPα antibodies or antigen bindingfragments of the invention are able to block binding of human SIRPα tohuman CD47. The ability to block binding of human SIRPα to human CD47can be determined using any method known in the art. In one embodiment,the ability of the antibodies to block binding of human SIRPα to humanCD47 is determined using an ELISA assay.

Methods of Making Antibodies and Antigen-binding Fragments Thereof

Thus, the present invention includes methods for making an anti-SIRPαantibody or antigen-binding fragment thereof of the present inventioncomprising culturing a hybridoma cell that expresses the antibody orfragment under condition favorable to such expression and, optionally,isolating the antibody or fragment from the hybridoma and/or the growthmedium (e.g. cell culture medium).

The anti-SIRPα antibodies disclosed herein may also be producedrecombinantly (e.g., in an E. coli/T7 expression system, a mammaliancell expression system or a lower eukaryote expression system). In thisembodiment, nucleic acids encoding the antibody immunoglobulin moleculesof the invention (e.g., V_(H) or V_(L)) may be inserted into a pET-basedplasmid and expressed in the E. coli/T7 system. For example, the presentinvention includes methods for expressing an antibody or antigen-bindingfragment thereof or immunoglobulin chain thereof in a host cell (e.g.,bacterial host cell such as E. coli such as BL21 or BL21DE3) comprisingexpressing T7 RNA polymerase in the cell which also includes apolynucleotide encoding an immunoglobulin chain that is operably linkedto a T7 promoter. For example, in an embodiment of the invention, abacterial host cell, such as a E. coli, includes a polynucleotideencoding the T7 RNA polymerase gene operably linked to a lac promoterand expression of the polymerase and the chain is induced by incubationof the host cell with IPTG (isopropyl-beta-D-thiogalactopyrano side).

There are several methods by which to produce recombinant antibodieswhich are known in the art. One example of a method for recombinantproduction of antibodies is disclosed in U.S. Pat. No. 4,816,567.

Transformation can be by any known method for introducingpolynucleotides into a host cell. Methods for introduction ofheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,biolistic injection and direct microinjection of the DNA into nuclei. Inaddition, nucleic acid molecules may be introduced into mammalian cellsby viral vectors. Methods of transforming cells are well known in theart. See, for example, U.S. Pat. Nos. 4,399,216; 4,912,040; 4,740,461and 4,959,455.

Thus, the present invention includes recombinant methods for making ananti-SIRPα antibody or antigen-binding fragment thereof of the presentinvention, or an immunoglobulin chain thereof, comprising introducing apolynucleotide encoding one or more immunoglobulin chains of theantibody or fragment (e.g., heavy and/or light immunoglobulin chain);culturing the host cell (e.g., CHO or Pichia or Pichia pastoris) undercondition favorable to such expression and, optionally, isolating theantibody or fragment or chain from the host cell and/or medium in whichthe host cell is grown.

Anti-SIRPα antibodies can also be synthesized by any of the methods setforth in U.S. Pat. No. 6,331,415.

Eukaryotic and prokaryotic host cells, including mammalian cells ashosts for expression of the antibodies or fragments or immunoglobulinchains disclosed herein are well known in the art and include manyimmortalized cell lines available from the American Type CultureCollection (ATCC). These include, inter alia, Chinese hamster ovary(CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number ofother cell lines. Mammalian host cells include human, mouse, rat, dog,monkey, pig, goat, bovine, horse and hamster cells. Cell lines ofparticular preference are selected through determining which cell lineshave high expression levels. Other cell lines that may be used areinsect cell lines, such as Sf9 cells, amphibian cells, bacterial cells,plant cells and fungal cells. Fungal cells include yeast and filamentousfungus cells including, for example, Pichia pastoris, Pichia finlandica,Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichiaminuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp.,Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporiumlucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum,Physcomitrella patens and Neurospora crassa. Pichia sp., anySaccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candidaalbicans, any Aspergillus sp., Trichoderma reesei, Chrysosporiumlucknowense, any Fusarium sp., Yarrowia lipolytica, and Neurosporacrassa. When recombinant expression vectors encoding the heavy chain orantigen-binding portion or fragment thereof, and/or the light chain orantigen-binding fragment thereof are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody orfragment or chain in the host cells or secretion into the culture mediumin which the host cells are grown.

Antibodies and antigen-binding fragments thereof and immunoglobulinchains can be recovered from the culture medium using standard proteinpurification methods. Further, expression of antibodies andantigen-binding fragments thereof and immunoglobulin chains of theinvention (or other moieties therefrom) from production cell lines canbe enhanced using a number of known techniques. For example, theglutamine synthetase gene expression system (the GS system) is a commonapproach for enhancing expression under certain conditions. The GSsystem is discussed in whole or part in connection with European PatentNos. 0216846, 0256055, and 0323997 and 0338841. Thus, in an embodimentof the invention, the mammalian host cells (e.g., CHO) lack a glutaminesynthetase gene and are grown in the absence of glutamine in the mediumwherein, however, the polynucleotide encoding the immunoglobulin chaincomprises a glutamine synthetase gene which complements the lack of thegene in the host cell.

The present invention includes methods for purifying an anti-SIRPαantibody or antigen-binding fragment thereof of the present inventioncomprising introducing a sample comprising the antibody or fragment to apurification medium (e.g., cation exchange medium, anion exchangemedium, hydrophobic exchange medium, affinity purification medium (e.g.,protein-A, protein-G, protein-A/G, protein-L)) and either collectingpurified antibody or fragment from the flow-through fraction of saidsample that does not bind to the medium; or, discarding the flow-throughfraction and eluting bound antibody or fragment from the medium andcollecting the eluate. In an embodiment of the invention, the medium isin a column to which the sample is applied. In an embodiment of theinvention, the purification method is conducted following recombinantexpression of the antibody or fragment in a host cell, e.g., wherein thehost cell is first lysed and, optionally, the lysate is purified ofinsoluble materials prior to purification on a medium.

In general, glycoproteins produced in a particular cell line ortransgenic animal will have a glycosylation pattern that ischaracteristic for glycoproteins produced in the cell line or transgenicanimal. Therefore, the particular glycosylation pattern of an antibodywill depend on the particular cell line or transgenic animal used toproduce the antibody. However, all antibodies encoded by the nucleicacid molecules provided herein, or comprising the amino acid sequencesprovided herein, comprise the instant invention, independent of theglycosylation pattern the antibodies may have. Similarly, in particularembodiments, antibodies with a glycosylation pattern comprising onlynon-fucosylated N-glycans may be advantageous, because these antibodieshave been shown to typically exhibit more potent efficacy than theirfucosylated counterparts both in vitro and in vivo (See for example,Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Pat. Nos.6,946,292 and 7,214,775). These antibodies with non-fucosylatedN-glycans are not likely to be immunogenic because their carbohydratestructures are a normal component of the population that exists in humanserum IgG.

The present invention includes bispecific and bifunctional antibodiesand antigen-binding fragments having a binding specificity for SIRPα andanother antigen such as, for example, CD19, CD20, CD22, CD24, CD25,CD30, CD33, CD38, CD44, CD52, CD56, CD70, CD96, CD97, CD99, CD117,CD123, c-Met, CEA, EGFR, EpCAM, HER2, HERS, PSMA, PTHR2, mesothelin,PD-1, PD-L1, TIM3, and methods of use thereof. A bispecific orbifunctional antibody is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai, et al., (1990) Clin. Exp. Immunol. 79: 315-321, Kostelny,et al., (1992) J Immunol. 148:1547-1553. In addition, bispecificantibodies may be formed as “diabodies” (Holliger, et al., (1993) PNASUSA 90:6444-6448) or as “Janusins” (Traunecker, et al., (1991) EMBO J.10:3655-3659 and Traunecker, et al., (1992) Int. J. Cancer Suppl.7:51-52). Included are “Duobodies,” which are bispecific antibodies withnormal IgG structures (Labrijn et al., 2013, Proc. Natl. Acad. Sci. USA110 (13): 5145-5150).

The present invention further includes anti-SIRPα antigen-bindingfragments of the anti-SIRPα antibodies disclosed herein. The antibodyfragments include F(ab)₂ fragments, which may be produced by enzymaticcleavage of an IgG by, for example, pepsin. Fab fragments may beproduced by, for example, reduction of F(ab)₂ with dithiothreitol ormercaptoethylamine.

Immunoglobulins may be assigned to different classes depending on theamino acid sequences of the constant domain of their heavy chains. Insome embodiments, different constant domains may be appended tohumanized V_(L) and V_(H) regions derived from the CDRs provided herein.There are at least five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3 and IgG4; IgA1 and IgA2. The inventioncomprises antibodies and antigen-binding fragments of any of theseclasses or subclasses of antibodies.

In one embodiment, the antibody or antigen-binding fragment comprises aheavy chain constant region, e.g. a human constant region, such as γ1,γ2, γ3, or γ4 human heavy chain constant region or a variant thereof. Inanother embodiment, the antibody or antigen-binding fragment comprises alight chain constant region, e.g. a human light chain constant region,such as lambda or kappa human light chain region or variant thereof. Byway of example, and not limitation the human heavy chain constant regioncan be γ4 and the human light chain constant region can be kappa. In analternative embodiment, the Fc region of the antibody is γ4 with aSer228Pro mutation (Schuurman, J et. al., Mol. Immunol. 38: 1-8, 2001).

In one embodiment, the antibody or antigen-binding fragment comprises aheavy chain constant region of the IgG1 subtype. In one embodiment, theantibody or antigen-binding fragment comprises a heavy chain constantregion of the IgG2 subtype. In one embodiment, the antibody orantigen-binding fragment comprises a heavy chain constant region of theIgG4 subtype.

Antibody Engineering

Further included are embodiments in which the anti-SIRPα antibodies andantigen-binding fragments thereof are engineered antibodies to includemodifications to framework residues within the variable domains theantibody, e.g. to improve the properties of the antibody or fragment.Typically, such framework modifications are made to decrease theimmunogenicity of the antibody or fragment. This is usually accomplishedby replacing non-CDR residues in the variable domains (i.e. frameworkresidues) in a parental (e.g. rodent) antibody or fragment withanalogous residues from the immune repertoire of the species in whichthe antibody is to be used, e.g. human residues in the case of humantherapeutics. Such an antibody or fragment is referred to as a“humanized” antibody or fragment. In some cases, it is desirable toincrease the affinity, or alter the specificity of an engineered (e.g.humanized) antibody. One approach is to mutate one or more frameworkresidues to the corresponding germline sequence. More specifically, anantibody or fragment that has undergone somatic mutation can containframework residues that differ from the germline sequence from which theantibody is derived. Such residues can be identified by comparing theantibody or fragment framework sequences to the germline sequences fromwhich the antibody or fragment is derived. Another approach is to revertto the original parental (e.g., rodent) residue at one or more positionsof the engineered (e.g. humanized) antibody, e.g. to restore bindingaffinity that may have been lost in the process of replacing theframework residues. (See, e.g., U.S. Pat. Nos. 5,693,762, 5,585,089 andU.S. Pat. No. 5,530,101).

In certain embodiments, the anti-SIRPα antibodies and antigen-bindingfragments thereof are engineered (e.g. humanized) to includemodifications in the framework and/or CDRs to improve their properties.Such engineered changes can be based on molecular modelling. A molecularmodel for the variable region for the parental (non-human) antibodysequence can be constructed to understand the structural features of theantibody and used to identify potential regions on the antibody that caninteract with the antigen. Conventional CDRs are based on alignment ofimmunoglobulin sequences and identifying variable regions. Kabat et al.,(1991) Sequences of Proteins of Immunological Interest, Kabat, et al.;National Institutes of Health, Bethesda, MD; 5^(th) ed.; NIH Publ. No.91-3242; Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J.Biol. Chem. 252:6609-6616. Chothia and coworkers carefully examinedconformations of the loops in crystal structures of antibodies andproposed hypervariable loops. Chothia, et al., (1987) J. Mol. Biol.196:901-917 or Chothia, et al., (1989) Nature 342:878-883. There arevariations between regions classified as “CDRs” and “hypervariableloops”. Later studies (Raghunathan et al, (2012) J. Mol Recog. 25, 3,103-113) analyzed several antibody—antigen crystal complexes andobserved that the antigen binding regions in antibodies do notnecessarily conform strictly to the “CDR” residues or “hypervariable”loops. The molecular model for the variable region of the non-humanantibody can be used to guide the selection of regions that canpotentially bind to the antigen. In practice the potential antigenbinding regions based on the model differ from the conventional “CDR”sor “hypervariable” loops. Commercial scientific software such asDiscovery Studio (BIOVIA, Dassault Systems)) can be used for molecularmodeling. Human frameworks can be selected based on best matches withthe non-human sequence both in the frameworks and in the CDRs. For FR4(framework 4) in VH, VJ regions for the human germlines are comparedwith the corresponding non-human region. In the case of FR4 (framework4) in VL, J-kappa and J-Lambda regions of human germline sequences arecompared with the corresponding non-human region. Once suitable humanframeworks are identified, the CDRs are grafted into the selected humanframeworks. In some cases, certain residues in the VL-VH interface canbe retained as in the non-human (parental) sequence. Molecular modelscan also be used for identifying residues that can potentially alter theCDR conformations and hence binding to antigen. In some cases, theseresidues are retained as in the non-human (parental) sequence. Molecularmodels can also be used to identify solvent exposed amino acids that canresult in unwanted effects such as glycosylation, deamidation andoxidation. Developability filters can be introduced early on in thedesign stage to eliminate/minimize these potential problems.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. Pat. No.7,125,689.

In particular embodiments, it will be desirable to change certain aminoacids containing exposed side-chains to another amino acid residue inorder to provide for greater chemical stability of the final antibody,so as to avoid deamidation or isomerization. The deamidation ofasparagine may occur on NG, DG, NG, NS, NA, NT, QG or QS sequences andresult in the creation of an isoaspartic acid residue that introduces akink into the polypeptide chain and decreases its stability (isoasparticacid effect). Isomerization can occur at DG, DS, DA or DT sequences. Incertain embodiments, the antibodies of the present disclosure do notcontain deamidation or asparagine isomerism sites.

For example, an asparagine (Asn) residue may be changed to Gln or Ala toreduce the potential for formation of isoaspartate at any Asn-Glysequences, particularly within a CDR. A similar problem may occur at aAsp-Gly sequence. Reissner and Aswad (2003) Cell. Mol. Life Sci.60:1281. Isoaspartate formation may debilitate or completely abrogatebinding of an antibody to its target antigen. See, Presta (2005) J.Allergy Clin. Immunol. 116:731 at 734. In one embodiment, the asparagineis changed to glutamine (Gln). It may also be desirable to alter anamino acid adjacent to an asparagine (Asn) or glutamine (Gln) residue toreduce the likelihood of deamidation, which occurs at greater rates whensmall amino acids occur adjacent to asparagine or glutamine. See,Bischoff & Kolbe (1994) J. Chromatog. 662:261. In addition, anymethionine residues (typically solvent exposed Met) in CDRs may bechanged to Lys, Leu, Ala, or Phe or other amino acids in order to reducethe possibility that the methionine sulfur would oxidize, which couldreduce antigen-binding affinity and also contribute to molecularheterogeneity in the final antibody preparation. Id. Additionally, inorder to prevent or minimize potential scissile Asn-Pro peptide bonds,it may be desirable to alter any Asn-Pro combinations found in a CDR toGln-Pro, Ala-Pro, or Asn-Ala. Antibodies with such substitutions aresubsequently screened to ensure that the substitutions do not decreasethe affinity or specificity of the antibody for SIRPα, or other desiredbiological activity to unacceptable levels.

TABLE 2 Exemplary stabilizing CDR variants CDR Residue StabilizingVariant Sequence Asn-Gly Gln-Gly, Ala-Gly, or Asn-Ala (N-G) (Q-G),(A-G), or (N-A) Asp-Gly Glu-Gly, Ala-Gly or Asp-Ala (D-G) (E-G), (A-G),or (D-A) Met Lys, Leu, Ala, or Phe (M) (K), (L), (A), or (F) Asn Gln orAla (N) (Q) or (A) Asn-Pro Gln-Pro, Ala-Pro, or Asn-Ala (N-P) (Q-P),(A-P), or (N-A)

Another type of framework modification involves mutating one or moreresidues within the framework regions to prevent aggregation. The riskof an antibody to aggregate can be assessed using the spatialaggregation propensity—See, Chennamsetty, N et al (2010) J. Phys. Chem.114, 6614-6624. The method requires the calculation of the SolventAccessible Area (SAA) for each atom. The molecular aggregation score isthen calculated as the sum of all atomic scores. For a given radius andsize of molecule, this is an approximate indication of its overalltendency to aggregate. Residues with a high aggregation score arereplaced by residues with a lower score (e.g. more hydrophilic aminoacids).

Antibody Engineering of the Fc region

The antibodies (e.g., humanized antibodies) and antigen-bindingfragments thereof disclosed herein can also be engineered to includemodifications within the Fc region, typically to alter one or moreproperties of the antibody, such as serum half-life, complementfixation, Fc receptor binding, and/or effector function (e.g.,antigen-dependent cellular cytotoxicity). Furthermore, the antibodiesand antigen-binding fragments thereof disclosed herein can be chemicallymodified (e.g., one or more chemical moieties can be attached to theantibody) or be modified to alter its glycosylation, again to alter oneor more properties of the antibody or fragment. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

The antibodies and antigen-binding fragments thereof disclosed hereinalso include antibodies and fragments with modified (or blocked) Fcregions to provide altered effector functions. See, e.g., U.S. Pat. No.5,624,821; WO2003/086310; WO2005/120571; WO2006/0057702. Suchmodifications can be used to enhance or suppress various reactions ofthe immune system, with possible beneficial effects in diagnosis andtherapy. Alterations of the Fc region include amino acid changes(substitutions, deletions and insertions), glycosylation ordeglycosylation, and adding multiple Fc regions. Changes to the Fc canalso alter the half-life of antibodies in therapeutic antibodies,enabling less frequent dosing and thus increased convenience anddecreased use of material. See Presta (2005) J. Allergy Clin. Immunol.116:731 at 734-35.

In one embodiment, the antibody or antigen-binding fragment of theinvention is an IgG4 isotype antibody or fragment comprising a Serine toProline mutation at a position corresponding to position 228 (S228P; EUindex; SEQ ID NO: 66) in the hinge region of the heavy chain constantregion. This mutation has been reported to abolish the heterogeneity ofinter-heavy chain disulfide bridges in the hinge region (Angal et al(1993). Mol. Immunol. 30:105-108; position 241 is based on the Kabatnumbering system).

In one embodiment of the invention, the hinge region of CH1 is modifiedsuch that the number of cysteine residues in the hinge region isincreased or decreased. This approach is described further in U.S. Pat.No. 5,677,425. The number of cysteine residues in the hinge region ofCH1 is altered, for example, to facilitate assembly of the light andheavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody orantigen-binding fragment of the invention is mutated to decrease thebiological half-life of the antibody or fragment. More specifically, oneor more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody orfragment has impaired Staphylococcyl protein A (SpA) binding relative tonative Fc-hinge domain SpA binding. This approach is described infurther detail in U.S. Pat. No. 6,165,745.

In another embodiment, the antibody or antigen-binding fragment of theinvention is modified to increase its biological half-life. Variousapproaches are possible. For example, one or more of the followingmutations can be introduced: T252L, T254S, T256F, as described in U.S.Pat. No. 6,277,375. Alternatively, to increase the biological half-life,the antibody can be altered within the CH1 or CL region to contain asalvage receptor binding epitope taken from two loops of a CH2 domain ofan Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and6,121,022.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody or antigen-binding fragment. Forexample, one or more amino acids selected from amino acid residues 234,235, 236, 237, 297, 318, 320 and 322 can be replaced with a differentamino acid residue such that the antibody has an altered affinity for aneffector ligand and retains the antigen-binding ability of the parentantibody. The effector ligand to which affinity is altered can be, forexample, an Fc receptor or the Cl component of complement. This approachis described in further detail in U.S. Pat. Nos. 5,624,821 and5,648,260.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351.

The proteins of the invention, which are preferably antibodies and mostpreferably IgG antibodies or fragments thereof, may have altered (e.g.,relative to an unmodified antibody) FcγR binding properties (examples ofbinding properties include but are not limited to, binding specificity,equilibrium dissociation constant (K_(D)), dissociation and associationrates (k_(off) and k_(on) respectively), binding affinity and/oravidity) and that certain alterations are more or less desirable. It isknown in the art that the equilibrium dissociation constant (K_(D)) isdefined as k_(off)/k_(on), and K_(a) is the reciprocal of K_(D).

The affinities and binding properties of an Fc region for its ligand,may be determined by a variety of in vitro assay methods (biochemical orimmunological based assays) known in the art for determining Fc-FcγRinteractions, i.e., specific binding of an Fc region to an FcγRincluding but not limited to, equilibrium methods (e.g., enzyme-linkedimmuno absorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics(e.g. BIACORE®, Octet®, or KinExa® analysis), and other methods such asindirect binding assays, competitive inhibition assays, fluorescenceresonance energy transfer (FRET), gel electrophoresis and chromatography(e.g., gel filtration). These and other methods may utilize a label onone or more of the components being examined and/or employ a variety ofdetection methods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels.

In certain embodiments, the proteins of the present invention bind toone or more human FcγRs selected from the group consisting of FcγRI,FcγRIIB, FcγRIIC, FcγRIIIA-F158, and FcγRIIIA-V158 with an affinity atleast 10-fold, preferably at least 30-fold, and more preferably at least100-fold, less than equivalent protein having a wild-type human IgG1heavy chain constant domain (SEQ ID NO: 119) Fc region or a wild-typehuman IgG4 heavy chain constant domain (SEQ ID NO: 66) Fc region.

In various embodiments, the proteins of the invention comprise animmunoglobulin Fc region comprising an immunoglobulin C2 region and animmunoglobulin C3 region and an immunoglobulin hinge region. By way ofexample, the immunoglobulin Fc region may be an IgG Fc region, an IgE Fcregion, or an IgA Fc region. In certain preferred embodiments, theprotein comprises two immunoglobulin Fc regions, each immunoglobulin Fcregion comprising an immunoglobulin C2 region and an immunoglobulin C3region and an immunoglobulin hinge region, wherein the hinge region ofone of the immunoglobulin Fc regions is bound to the hinge region of theother immunoglobulin Fc region to form a dimeric Fc structure. Mostpreferably, such a protein is a human or humanized IgG protein.

In certain embodiments, the proteins of the invention comprise a mutatedIgG4 Fc region, and preferably the protein is an IgG comprising twomutated IgG4 Fc regions to form a dimeric Fc structure. By way ofexample, a mutated IgG4 Fc region may comprise one of the mutations, ormutational combinations, recited in Table 3. The numbering system of theconstant region referred to in this table is that of the EU index as setforth in Kabat et al. (1991, NIH Publication 91-3242, National TechnicalInformation Service, Springfield, Va.). In the table, the first letterand number represent the unmodified amino acid and its position and thesecond letter represents the substituted amino acid at said position.For those entries that include combinations of more than one mutation,each mutation in the combination is separated by a “/”.

TABLE 3 N297Q L235E N297Q/L235E F234A Q268A F234A/L235A/G237A/ P238AF234A/L235A/ΔG236/ F234A/L235A/G237A/ F234A/L235A/ΔG236/ G237A/P238AP238A/Q268A G237A/P238A/Q268A F234A/L235A L235E/P329G L235A/G237A/E318AF234A/L235A/G237A/ F234A/L235A/ΔG236/ F234A/L235A/G237A/ P238SG237A/P238S P238S/Q268A F234A/L235A/ΔG236/ G237A/P238S/Q268A

In certain embodiments, the proteins of the invention comprise a mutatedIgG1 Fc region, and preferably the protein is an IgG comprising twomutated IgG1 Fc regions to form a dimeric Fc structure. By way ofexample, a mutated IgG1 Fc region may comprise one of the mutationsrecited in Table 4. The numbering system of the constant region referredto in this table is that of the EU index as set forth in Kabat et al.(1991, NIH Publication 91-3242, National Technical Information Service,Springfield, VA). In the table, the first letter and number representthe unmodified amino acid and its position and the second letterrepresents the substituted amino acid at said position.

TABLE 4 K222Y P232K A231K E233N E233Q E233R E233S E233T E233H E233AE233V E233L E233F E233M E233Y E233W E233G L234D L234E L234N L234Q L234TL234H L234F L234K L234R L234S L234A L234M L234V L235E L235T L235F L235KL235R L235A L235M L235W L235N L235Q L235H L235V G236A G236N G236R G236HG236L G236F G236P G237A G237E G237N G237Q G237K G237R G237S G237T G237HG237L G237I G237F G237M G237Y G237P P238K P238N P238R P238S P238T P238YP238G P238A S239A S239N S239F S239K S239R S239V S239W S239P S239H S239YD249H V240A F241W F241L F243W F243L F243E P244H P245A P247V P247G V253IV263I V263T V263M V264D V264E V264K V264F V264M V264H V264W V264G V264QV264A V264L D265A D265E D265Q D265S D265H D265V D265L D265F D265M D265YD265N D265G V266T V266M V266A S267G S267H S267N S267P S267R S267T S267FS267W E269A E269K E269S E269V E269F E269I E269M E269W E269H E269T E269LE269N E269Y E269R E269P E269G D270A D270N D270E D270Q D270T D270H D270RD270S D270L D270I D270F D270W D270P D270G P271H P271Q P271K P271R P271SP271V P271F P271W D280L D280W D280P E293F E294A E293Y E294K E294R E294SE294V E294L E294F Q295A Q295W Q295P Q295G Y296E Y296Q Y296D Y296N Y296SY296T Y296L Y296I Y296A Y296V Y296M N297S N297D N297Q N297A S298T S298NS298K S298R T299A T299H T299D T299E T299N T299Q T299K T299R T299I T299FT299M T299Y T299W T299S T299V T299P T299G Y300E Y300K Y300R Y300S Y300PY300W V303A V303D W313F E318A E318V E318Q E318H E318L E318Y K320A K322AK322E N325A N325V N325H N325K N325Y N325W N325P N325G N325Q N325D N325EN325L N325I A327Q A327E A327N A327L A327I A327F A327W L328N L328F L328HL328R L328T L328V L328I L328P L328M L328E L328A P329A P329F P329D P329NP329Q P329K P329S P329T P329H P329V P329L P329M P329Y P329W P329G P329RA330L A330R A330P A330T A330V A330F A330H P331A P331S P331N P331E I332KI332N I332Q I332T I332H I332Y I332A I332R E333N E333R I336E I336Y S337H

In certain embodiments, a mutated IgG1 Fc region may comprise one of themutational combinations recited in Table 5. The numbering system of theconstant region referred to in this table is that of the EU index as setforth in Kabat et al. (1991, NIH Publication 91-3242, National TechnicalInformation Service, Springfield, Va.). In the table, the first letterand number represent the unmodified amino acid and its position and thesecond letter represents the substituted amino acid at said position.For each of the combinations of more than one mutation, each mutation inthe combination is separated by a “/” and deletions are indicated by a“Δ”.

TABLE 5 C220S/C226S/C229S/P238S C226S/C229S/E233P/L234V/E233P/L234V/L235A L235A E233P/L234V/L235A/ΔG236 E233P/L234V/L235A/ΔG236/L234A/L235A A327G/A330S/P331S L235A/G237A L235A/G237A/E318S/K320S/L235A/G237A/P331A K322S L234F/L235E L234F/L235E/D265A L234F/L235E/D265A/N297Q/P331S L234F/L235E/N297Q L234F/L235E/P329G L234F/L235A/K322Q/M252Y/S254T/T256E L234F/L235Q/K322Q/M252Y/ L234F/L235Q/P331G/M252Y/G236R/L328R S254T/T256E S254T/T256E S239D/D265I/N297D/I332ES239D/D265L/N297D/I332E S239D/D265F/N297D/ I332E S239D/D265Y/N297D/I332ES239D/D265T/N297D/I332E S239D/N297D/A330Y/ I332ES239D/F241S/F243H/V262T/ V264E/N297D/I332E D265A/P331SV264T/N297D/K326E/I332E D265A/N297Q N297D/D265Y/T299L/I332EN297D/D265Y/I332E N297D/I332E/Y296D N297D/I332E N297D/I332E/Y296EN297D/I332E/Y296N N297D/I332E/Y296Q N297D/I332E/Y296H N297D/I332E/Y296TN297D/I332E/T299V N297D/I332E/T299I N297D/I332E/T299L N297D/I332E/T299FN297D/I332E/T299H N297D/I332E/T299E N297D/1332E/A330Y N297D/I332E/S298A/A330Y N297E/D265F/I332E N297E/I332E F241E/F243R/V262E/ V264RF241E/F243Q/V262T/V264E F241L/F243L/V262I/V264I F241W/F243WF241W/F243W/V262A/V264A F241L/V262I F243L/V262I/V264WF241Y/F243Y/V262T/V264T F241E/F243R/V262E/V264R F241E/F243Q/V262T/V264EF241R/F243Q/V262T/V264R F241E/F243Y/V262T/V264R P244H/P245A/P247VF241E/F243R/V262E/V264R/ F241E/F243Y/V262T/V264R F241E/F243Y/V262T/I332E V264R/I332E S239E/D265G S239E/D265N S239E/D265Q M252Y/S254T/T256ES267Q/A327S S267L/A327S N297S/I332E S239N/I332N S239N/I332Q S239Q/I332NS239Q/I332Q S298N/Y300S S298N/T299A/Y300S N297Q/S298N/Y300SE318S/K320S/K322S E318S/K320S/K322S/P311A L328E/I332E L328N/I332EL234A/L235A/G237A/P238A/ L234A/L235A/G237A/P238S/L234A/L235A/G237A/P238A/ H268A/A330S/P331S H268A/A330S/P331SH268A/A330S/P331S L328Q/I332E L328H/I332E

In certain embodiments, the proteins of the invention comprise a wildtype or mutated IgG2 Fc region, and preferably the protein is an IgGcomprising two wild type or mutated IgG2 Fc regions to form a dimeric Fcstructure. A mutated IgG2 Fc region may comprise one of the mutations,or mutational combinations, recited in Table 6. The numbering system ofthe constant region referred to in this table is that of the EU index asset forth in Kabat et al. (1991, NIH Publication 91-3242, NationalTechnical Information Service, Springfield, Va.). In the table, thefirst letter and number represent the unmodified amino acid and itsposition and the second letter represents the substituted amino acid atsaid position. For those entries that include combinations of more thanone mutation, each mutation in the combination is separated by a “/”.

TABLE 6 V234A G237A A235E/G237A V234A/A235E/G237A V234A/G237AV234A/G237A/P238S H268Q/V309L/A330S/P331S V234A/G237A/H268A/V309L/V234A/G237A/H268Q/V309L/ A330S/P331S A330S/P331SV234A/G237A/P238S/H268A/ P233S/V234A/G237A/P238SP233S/V234A/G237A/H268A/ V309L/A330S/P331S V309L/A330S/P331SP233S/V234A/G237A/H268Q/ P233S/V234A/G237A/P238S/ V309L/A330S/P331SH268A/V309L/A330S/P331S

Production of Antibodies with Modified Glycosylation

In still another embodiment, the antibodies or antigen-binding fragmentsof the invention comprise a particular glycosylation pattern. Forexample, an afucosylated or an aglycosylated antibody or fragment can bemade (i.e., the antibody lacks fucose or glycosylation, respectively).The glycosylation pattern of an antibody or fragment may be altered to,for example, increase the affinity or avidity of the antibody orfragment for a SIRPα antigen. Such modifications can be accomplished by,for example, altering one or more of the glycosylation sites within theantibody or fragment sequence. For example, one or more amino acidsubstitutions can be made that result in removal of one or more of thevariable region framework glycosylation sites to thereby eliminateglycosylation at that site. Such deglycosylation may increase theaffinity or avidity of the antibody or fragment for antigen. See, e.g.,U.S. Pat. Nos. 5,714,350 and 6,350,861.

Antibodies and antigen-binding fragments disclosed herein may furtherinclude those produced in lower eukaryote host cells, in particularfungal host cells such as yeast and filamentous fungi have beengenetically engineered to produce glycoproteins that have mammalian- orhuman-like glycosylation patterns (See for example, Choi et al, (2003)Proc. Natl. Acad. Sci. 100: 5022-5027; Hamilton et al., (2003) Science301: 1244-1246; Hamilton et al., (2006) Science 313: 1441-1443; Nett etal., Yeast 28(3):237-52 (2011); Hamilton et al., Curr Opin Biotechnol.18(5): 387-92 (2007)). A particular advantage of these geneticallymodified host cells over currently used mammalian cell lines is theability to control the glycosylation profile of glycoproteins that areproduced in the cells such that compositions of glycoproteins can beproduced wherein a particular N-glycan structure predominates (see,e.g., U.S. Pat. Nos. 7,029,872 and 7,449,308). These geneticallymodified host cells have been used to produce antibodies that havepredominantly particular N-glycan structures (See for example, Li etal., (2006) Nat. Biotechnol. 24: 210-215).

In particular embodiments, the antibodies and antigen-binding fragmentsthereof disclosed herein further include those produced in lowereukaryotic host cells and which comprise fucosylated and non-fucosylatedhybrid and complex N-glycans, including bisected and multiantennaryspecies, including but not limited to N-glycans such asGlcNAc₍₁₋₄₎Man₃GlcNAc₂; Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂;NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂.

In particular embodiments, the antibodies and antigen-binding fragmentsthereof provided herein may comprise antibodies or fragments having atleast one hybrid N-glycan selected from the group consisting ofGlcNAcMan₅GlcNAc₂; GalGlcNAcMan₅GlcNAc₂; and NANAGalGlcNAcMan₅GlcNAc₂.In particular aspects, the hybrid N-glycan is the predominant N-glycanspecies in the composition.

In particular embodiments, the antibodies and antigen-binding fragmentsthereof provided herein comprise antibodies and fragments having atleast one complex N-glycan selected from the group consisting ofGlcNAcMan₃GlcNAc₂; GalGlcNAcMan₃GlcNAc₂; NANAGalGlcNAcMan₃GlcNAc₂;GlcNAc₂Man₃GlcNAc₂; GalGlcNAc₂Man₃GlcNAc₂; Gal₂GlcNAc₂Man₃GlcNAc₂;NANAGal₂GlcNAc₂Man₃GlcNAc₂; and NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. Inparticular aspects, the complex N-glycan are the predominant N-glycanspecies in the composition. In further aspects, the complex N-glycan isa particular N-glycan species that comprises about 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N-glycans inthe composition. In one embodiment, the antibody and antigen bindingfragments thereof provided herein comprise complex N-glycans, wherein atleast 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of thecomplex N-glycans comprise the structure NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂,wherein such structure is afucosylated. Such structures can be produced,e.g., in engineered Pichia pastoris host cells.

In particular embodiments, the N-glycan is fucosylated. In general, thefucose is in an α1,3-linkage with the GlcNAc at the reducing end of theN-glycan, an α1,6-linkage with the GlcNAc at the reducing end of theN-glycan, an α1,2-linkage with the Gal at the non-reducing end of theN-glycan, an α1,3-linkage with the GlcNac at the non-reducing end of theN-glycan, or an α1,4-linkage with a GlcNAc at the non-reducing end ofthe N-glycan.

Therefore, in particular aspects of the above the glycoproteincompositions, the glycoform is in an α1,3-linkage or α1,6-linkage fucoseto produce a glycoform selected from the group consisting ofMan₅GlcNAc₂(Fuc), GlcNAcMan₅GlcNAc₂(Fuc), Man₃GlcNAc₂(Fuc),GlcNAcMan₃GlcNAc₂(Fuc), GlcNAc₂Man₃GlcNAc₂(Fuc),GalGlcNAc₂Man₃GlcNAc₂(Fuc), Gal₂GlcNAc₂Man₃GlcNAc₂(Fuc),NANAGal₂GlcNAc₂Man₃GlcNAc₂(Fuc), and NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂(Fuc);in an α1,3-linkage or α1,4-linkage fucose to produce a glycoformselected from the group consisting of GlcNAc(Fuc)Man₅GlcNAc₂,GlcNAc(Fuc)Man₃GlcNAc₂, GlcNAc₂(Fuc₁₋₂)Man₃GlcNAc₂,GalGlcNAc₂(Fuc₁₋₂)Man₃GlcNAc₂, Gal₂GlcNAc₂(Fuc₁₋₂)Man₃GlcNAc₂,NANAGal₂GlcNAc₂(Fuc₁₋₂)Man₃GlcNAc₂, andNANA₂Gal₂GlcNAc₂(Fuc₁₋₂)Man₃GlcNAc₂; or in an a1,2-linkage fucose toproduce a glycoform selected from the group consisting ofGal(Fuc)GlcNAc₂Man₃GlcNAc₂, Gal₂(Fuc₁₋₂)GlcNAc₂Man₃GlcNAc₂,NANAGal₂(Fuc₁₋₂)GlcNAc₂Man₃GlcNAc₂, andNANA₂Gal₂(Fuc₁₋₂)GlcNAc₂Man₃GlcNAc₂.

In further aspects, the antibodies (e.g., humanized antibodies) orantigen-binding fragments thereof comprise high mannose N-glycans,including but not limited to, Man₈GlcNAc₂, Man₇GlcNAc₂, Man₆GlcNAc₂,Man₅GlcNAc₂, Man₄GlcNAc₂, or N-glycans that consist of the Man₃GlcNAc₂N-glycan structure.

In further aspects of the above, the complex N-glycans further includefucosylated and non-fucosylated bisected and multiantennary species.

As used herein, the terms “N-glycan” and “glycoform” are usedinterchangeably and refer to an N-linked oligosaccharide, for example,one that is attached by an asparagine-N-acetylglucosamine linkage to anasparagine residue of a polypeptide. N-linked glycoproteins contain anN-acetylglucosamine residue linked to the amide nitrogen of anasparagine residue in the protein. The predominant sugars found onglycoproteins are glucose, galactose, mannose, fucose,N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialicacid (e.g., N-acetyl-neuraminic acid (NANA)). The processing of thesugar groups occurs co-translationally in the lumen of the ER andcontinues post-translationally in the Golgi apparatus for N-linkedglycoproteins.

N-glycans have a common pentasaccharide core of Man₃GlcNAc₂ (“Man”refers to mannose; “Glc” refers to glucose; and “NAc” refers toN-acetyl; GlcNAc refers to N-acetylglucosamine). Usually, N-glycanstructures are presented with the non-reducing end to the left and thereducing end to the right. The reducing end of the N-glycan is the endthat is attached to the Asn residue comprising the glycosylation site onthe protein. N-glycans differ with respect to the number of branches(antennae) comprising peripheral sugars (e.g., GlcNAc, galactose, fucoseand sialic acid) that are added to the Man₃GlcNAc₂ (“Man3”) corestructure which is also referred to as the “trimannose core”, the“pentasaccharide core” or the “paucimannose core”. N-glycans areclassified according to their branched constituents (e.g., high mannose,complex or hybrid). A “high mannose” type N-glycan has five or moremannose residues. A “complex” type N-glycan typically has at least oneGlcNAc attached to the 1,3 mannose arm and at least one GlcNAc attachedto the 1,6 mannose arm of a “trimannose” core. Complex N-glycans mayalso have galactose (“Gal”) or N-acetylgalactosamine (“GalNAc”) residuesthat are optionally modified with sialic acid or derivatives (e.g.,“NANA” or “NeuAc”, where “Neu” refers to neuraminic acid and “Ac” refersto acetyl). Complex N-glycans may also have intrachain substitutionscomprising “bisecting” GlcNAc and core fucose (“Fuc”). Complex N-glycansmay also have multiple antennae on the “trimannose core,” often referredto as “multiple antennary glycans.” A “hybrid” N-glycan has at least oneGlcNAc on the terminal of the 1,3 mannose arm of the trimannose core andzero or more mannoses on the 1,6 mannose arm of the trimannose core. Thevarious N-glycans are also referred to as “glycoforms.”

With respect to complex N-glycans, the terms “G-2”, “G-1”, “G0”, “G1”,“G2”, “A1”, and “A2” mean the following. “G-2” refers to an N-glycanstructure that can be characterized as Man₃GlcNAc₂; the term “G-1”refers to an N-glycan structure that can be characterized asGlcNAcMan₃GlcNAc₂; the term “G0” refers to an N-glycan structure thatcan be characterized as GlcNAc₂Man₃GlcNAc₂; the term “G1” refers to anN-glycan structure that can be characterized as GalGlcNAc₂Man₃GlcNAc₂;the term “G2” refers to an N-glycan structure that can be characterizedas Gal₂GlcNAc₂Man₃GlcNAc₂; the term “Al” refers to an N-glycan structurethat can be characterized as NANAGal₂GlcNAc₂Man₃GlcNAc₂; and, the term“A2” refers to an N-glycan structure that can be characterized asNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. Unless otherwise indicated, the terms G-2″,“G-1”, “G0”, “G1”, “G2”, “A1”, and “A2” refer to N-glycan species thatlack fucose attached to the GlcNAc residue at the reducing end of theN-glycan. When the term includes an “F”, the “F” indicates that theN-glycan species contains a fucose residue on the GlcNAc residue at thereducing end of the N-glycan. For example, G0F, G1F, G2F, A1F, and A2Fall indicate that the N-glycan further includes a fucose residueattached to the GlcNAc residue at the reducing end of the N-glycan.Lower eukaryotes such as yeast and filamentous fungi do not normallyproduce N-glycans that produce fucose.

With respect to multiantennary N-glycans, the term “multiantennaryN-glycan” refers to N-glycans that further comprise a GlcNAc residue onthe mannose residue comprising the non-reducing end of the 1,6 arm orthe 1,3 arm of the N-glycan or a GlcNAc residue on each of the mannoseresidues comprising the non-reducing end of the 1,6 arm and the 1,3 armof the N-glycan. Thus, multiantennary N-glycans can be characterized bythe formulas GlcNAc₍₂₋₄₎Man₃GlcNAc₂, Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂, orNANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. The term “1-4” refers to 1, 2,3, or 4 residues.

With respect to bisected N-glycans, the term “bisected N-glycan” refersto N-glycans in which a GlcNAc residue is linked to the mannose residueat the reducing end of the N-glycan. A bisected N-glycan can becharacterized by the formula GlcNAc₃Man₃GlcNAc₂ wherein each mannoseresidue is linked at its non-reducing end to a GlcNAc residue. Incontrast, when a multiantennary N-glycan is characterized asGlcNAc₃Man₃GlcNAc₂, the formula indicates that two GlcNAc residues arelinked to the mannose residue at the non-reducing end of one of the twoarms of the N-glycans and one GlcNAc residue is linked to the mannoseresidue at the non-reducing end of the other arm of the N-glycan.

In certain embodiments, the proteins of the invention comprise anaglycosylated Fc region. By way of example, an IgG1 Fc region may beaglycosylayed by deleting or substituting residue N297.

Antibody Physical Properties

The antibodies and antigen-binding fragments thereof disclosed hereinmay further contain one or more glycosylation sites in either the lightor heavy chain immunoglobulin variable region. Such glycosylation sitesmay result in increased immunogenicity of the antibody or fragment or analteration of the pK of the antibody due to altered antigen-binding(Marshall et al. (1972) Annu Rev Biochem 41:673-702; Gala and Morrison(2004) J Immunol 172:5489-94; Wallick et al (1988) J Exp Med168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985)Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).Glycosylation has been known to occur at motifs containing an N-X-S/Tsequence.

Each antibody or antigen-binding fragment will have a unique isoelectricpoint (pI), which generally falls in the pH range between 6 and 9.5. ThepI for an IgG1 antibody typically falls within the pH range of 7-9.5 andthe pI for an IgG4 antibody typically falls within the pH range of 6-8.

Each antibody or antigen-binding fragment will have a characteristicmelting temperature, with a higher melting temperature indicatinggreater overall stability in vivo (Krishnamurthy R and Manning M C(2002) Curr Pharm Biotechnol 3:361-71). In general, the T_(M1) (thetemperature of initial unfolding) may be greater than 60° C., greaterthan 65° C., or greater than 70° C. The melting point of an antibody orfragment can be measured using differential scanning calorimetry (Chenet al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett68:47-52) or circular dichroism (Murray et al. (2002) J. Chromatogr Sci40:343-9).

In a further embodiment, antibodies and antigen-binding fragmentsthereof are selected that do not degrade rapidly. Degradation of anantibody or fragment can be measured using capillary electrophoresis(CE) and MALDI-MS (Alexander A J and Hughes D E (1995) Anal Chem67:3626-32).

In a further embodiment, antibodies and antigen-binding fragmentsthereof are selected that have minimal aggregation effects, which canlead to the triggering of an unwanted immune response and/or altered orunfavorable pharmacokinetic properties. Generally, antibodies andfragments are acceptable with aggregation of 25% or less, 20% or less,15% or less, 10% or less, or 5% or less. Aggregation can be measured byseveral techniques, including size-exclusion column (SEC), highperformance liquid chromatography (HPLC), and light scattering.

Antibody Conjugates

The anti-SIRPα antibodies and antigen-binding fragments thereofdisclosed herein may also be conjugated to a chemical moiety. Thechemical moiety may be, inter alia, a polymer, a radionucleotide or acytotoxic factor. In particular embodiments, the chemical moiety is apolymer which increases the half-life of the antibody or fragment in thebody of a subject. Suitable polymers include, but are not limited to,hydrophilic polymers which include but are not limited to polyethyleneglycol (PEG) (e.g., PEG with a molecular weight of 2kDa, 5 kDa, 10 kDa,12kDa, 20 kDa, 30kDa or 40kDa), dextran and monomethoxypolyethyleneglycol (mPEG). Lee, et al., (1999) (Bioconj. Chem. 10:973-981) disclosesPEG conjugated single-chain antibodies. Wen, et al., (2001) (Bioconj.Chem. 12:545-553) disclose conjugating antibodies with PEG which isattached to a radiometal chelator (diethylenetriaminpentaacetic acid(DTPA)).

The antibodies and antigen-binding fragments thereof disclosed hereinmay also be conjugated with labels such as ⁹⁹Tc, ⁹⁰Y, ¹¹¹In, ³²P, ¹⁴C,¹²⁵I, ³H, ¹³¹I, ¹¹C, ¹⁵O, ¹³N, ¹⁸F, ³⁵S, ⁵¹Cr, ⁵⁷To, ²²⁶Ra, ⁶⁰Co, ₅₉Fe,⁵⁷Se, ¹⁵²Eu, ⁶⁷CU, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, ²³⁴Th, and ⁴⁰K,¹⁵⁷Gd, ⁵⁵Mn, ⁵²Tr, and ⁵⁶Fe.

The antibodies and antigen-binding fragments disclosed herein may alsobe PEGylated, for example to increase its biological (e.g., serum)half-life. To PEGylate an antibody or fragment, the antibody orfragment, typically is reacted with a reactive form of polyethyleneglycol (PEG), such as a reactive ester or aldehyde derivative of PEG,under conditions in which one or more PEG groups become attached to theantibody or antibody fragment. In particular embodiments, the PEGylationis carried out via an acylation reaction or an alkylation reaction witha reactive PEG molecule (or an analogous reactive water-solublepolymer). As used herein, the term “polyethylene glycol” is intended toencompass any of the forms of PEG that have been used to derivatizeother proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethyleneglycol or polyethylene glycol-maleimide. In certain embodiments, theantibody or fragment to be PEGylated is an aglycosylated antibody orfragment. Methods for PEGylating proteins are known in the art and canbe applied to the antibodies of the invention. See, e.g., EP 0 154 316and EP 0 401 384.

The antibodies and antigen-binding fragments disclosed herein may alsobe conjugated with fluorescent or chemilluminescent labels, includingfluorophores such as rare earth chelates, fluorescein and itsderivatives, rhodamine and its derivatives, isothiocyanate,phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde,fluorescamine, ¹⁵²Eu, dansyl, umbelliferone, luciferin, luminal label,isoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridimium salt label, an oxalate ester label, an aequorinlabel, 2,3-dihydrophthalazinediones, biotin/avidin, spin labels andstable free radicals.

The antibodies and antigen-binding fragments thereof of the inventionmay also be conjugated to a cytotoxic factor such as diptheria toxin,Pseudomonas aeruginosa exotoxin A chain , ricin A chain, abrin A chain,modeccin A chain, alpha-sarcin, Aleurites fordii proteins and compounds(e.g., fatty acids), dianthin proteins, Phytoiacca americana proteinsPAPI, PAPII, and PAP-S, Momordica charantia inhibitor, curcin, crotin,Saponaria officinalis inhibitor, mitogellin, restrictocin, phenomycin,and enomycin.

Any method known in the art for conjugating the antibodies andantigen-binding fragments thereof of the invention to the variousmoieties may be employed, including those methods described by Hunter,et al., (1962) Nature 144:945; David, et al., (1974) Biochemistry13:1014; Pain, et al., (1981) J. Immunol. Meth. 40:219; and Nygren, J.,(1982) Histochem. and Cytochem. 30:407. Methods for conjugatingantibodies and fragments are conventional and very well known in theart.

Therapeutic Uses of Anti-SIRPα Antibodies

Further provided are methods for treating subjects, including humansubjects, in need of treatment with the isolated antibodies orantigen-binding fragments thereof disclosed herein. In one embodiment ofthe invention, such subject suffers from an infection or an infectiousdisease.

In another embodiment of the invention, such subject suffers fromcancer. In one embodiment the cancer is, e.g., osteosarcoma,rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, renaltransitional cell cancer, bladder cancer, Wilm's cancer, ovarian cancer,pancreatic cancer, breast cancer, prostate cancer, bone cancer, lungcancer (e.g., non-small cell lung cancer), gastric cancer, colorectalcancer, cervical cancer, synovial sarcoma, head and neck cancer,squamous cell carcinoma, multiple myeloma, renal cell cancer,retinoblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma,rhabdoid tumor of the kidney, Ewing's sarcoma, chondrosarcoma, braincancer, glioblastoma, meningioma, pituitary adenoma, vestibularschwannoma, a primitive neuroectodermal tumor, medulloblastoma,astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma,choroid plexus papilloma, polycythemia vera, thrombocythemia, idiopathicmyelfibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer,carcinoid cancer or liver cancer, breast cancer or gastric cancer. In anembodiment of the invention, the cancer is metastatic cancer, e.g., ofthe varieties described above.

Cancers that may be treated by the antibodies or antigen-bindingfragments, compositions and methods of the invention include, but arenot limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma,rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma andteratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiatedsmall cell, undifferentiated large cell, adenocarcinoma), alveolar(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus(squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma),stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductaladenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors,vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors,Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma,fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma,hamartoma, leiomyoma) colorectal; Genitourinary tract: kidney(adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia),bladder and urethra (squamous cell carcinoma, transitional cellcarcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis(seminoma, teratoma, embryonal carcinoma, teratocarcinoma,choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),fallopian tubes (carcinoma), breast; Hematologic: blood (myeloidleukemia [acute and chronic], acute lymphoblastic leukemia, chroniclymphocytic leukemia, myeloproliferative diseases, multiple myeloma,myelodysplastic syndrome), Hodgkin's disease, non Hodgkin's lymphoma[malignant lymphoma]; Skin: malignant melanoma, basal cell carcinoma,squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi,lipoma, angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands:neuroblastoma. Thus, the term “cancerous cell” as provided herein,includes a cell afflicted by any one of the above-identified conditions.

In one embodiment, cancers that may be treated by the antibodies orantigen-binding fragments thereof disclosed herein, compositions andmethods of the invention include, but are not limited to: breast cancer,gastric cancer, esophageal cancer, gastroesophageal junction carcinoma,colorectal cancer, head and neck cancer, non-small cell lung cancer,osteosarcoma, neuroblastoma, bladder cancer, cervical cancer,endometrial cancer, ovarian cancer, lung cancer, squamous cellcarcinoma, melanoma, pancreatic cancer, prostate cancer, small cell lungcancer, kidney cancer, renal cell carcinoma, thyroid cancer,glioblastoma multiforme, fallopian tube cancer, peritoneal cancer,angiosarcoma, hepatocellular carcinoma, choriocarcinoma, soft tissuesarcoma, chronic lymphocytic leukemia, chronic myelocytic leukemia,non-Hodgkin's lymphoma, B-cell non-hodgkin's lymphoma, diffuse largeB-cell lymphoma, follicular lymphoma, mantle cell lymphoma,myelodysplastic syndrome, acute myelocytic leukemia, T-cell lymphoma,natural killer cell lymphoma, extranodal marginal zone B-cell lymphoma,acute lymphocytic leukemia, multiple myeloma.

In one embodiment, the antibodies or antigen-binding fragments thereofdisclosed herein may be used for the treatment of infections andinfectious diseases. As used herein, the term “infection” refers to anystate in at least one cell of an organism (i.e., a subject) is infectedby an infectious agent (e.g., a subject has an intracellular pathogeninfection, e.g., a chronic intracellular pathogen infection). As usedherein, the term “infectious agent” refers to a foreign biologicalentity (i.e. a pathogen) that induces CD47 expression (e.g., increasedCD47 expression) in at least one cell of the infected organism. Forexample, infectious agents include, but are not limited to bacteria,viruses, protozoans, and fungi.

Intracellular pathogens are of particular interest. Infectious diseasesare disorders caused by infectious agents. Some infectious agents causeno recognizable symptoms or disease under certain conditions, but havethe potential to cause symptoms or disease under changed conditions. Thesubject methods can be used in the treatment of chronic pathogeninfections, for example including but not limited to viral infections,e.g. retrovirus, lentivirus, hepadna virus, herpes viruses, pox viruses,human papilloma viruses, etc.; intracellular bacterial infections, e.g.Mycobacterium, Chlamydophila, Ehrlichia, Rickettsia, Brucella,Legionella, Francisella, Listeria, Coxiella, Neisseria, Salmonella,Yersinia sp, Helicobacter pylori etc.; and intracellular protozoanpathogens, e.g. Plasmodium sp, Trypanosoma sp., Giardia sp., Toxoplasmasp., Leishmania sp., etc.

In an embodiment, the invention provides methods for treating subjectsusing an anti-SIRPα antibody or antigen-binding fragment thereof of theinvention, wherein the subject suffers from a viral infection. In oneembodiment, the viral infection is an infection with a virus selectedfrom the group consisting of human immunodeficiency virus (HIV),hepatitis virus (A, B, or C), herpes virus (e.g., VZV, HSV-I, HAV-6,HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus,flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus,respiratory syncytial virus, mumps virus, rotavirus, measles virus,rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus,papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus orarboviral encephalitis virus.

In an embodiment, the invention provides methods for treating subjectsusing an anti-SIRPα antibody or antigen-binding fragment thereof of theinvention, wherein the subject suffers from a bacterial infection. Inone embodiment, the bacterial infection is infection with a bacteriaselected from the group consisting of Chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand gonococci, klebsiella, proteus, serratia, pseudomonas, Legionella,Corynebacterium diphtheriae, Salmonella, bacilli, Vibrio cholerae,Clostridium tetan, Clostridium botulinum, Bacillus anthricis, Yersiniapestis, Mycobacterium leprae, Mycobacterium lepromatosis, and Borriella.

In an embodiment, the invention provides methods for treating subjectsusing an anti-SIRPα antibody or antigen-binding fragment thereof of theinvention, wherein the subject suffers from a fungal infection. In oneembodiment, the fungal infection is an infection with a fungus selectedfrom the group consisting of Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

In an embodiment, the invention provides methods for treating subjectsusing an anti-SIRPα antibody or antigen-binding fragment thereof of theinvention, wherein the subject suffers from a parasitic infection. Inone embodiment, the parasitic infection is infection with a parasiteselected from the group consisting of Entamoeba histolytica, Balantidiumcoli, Naegleria fowleri, Acanthamoeba, Giardia lambia, Cryptosporidium,Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosomabrucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii andNippostrongylus brasiliensis.

A “subject” may be a mammal such as a human, dog, cat, horse, cow,mouse, rat, monkey (e.g., cynomolgous monkey, e.g., Macaca fascicularis)or rabbit. In preferred embodiments of the invention, the subject is ahuman subject.

The term “in association with” indicates that the componentsadministered in a method of the present invention (e.g., an anti-SIRPαantibody (e.g., humanized antibody) or antigen-binding fragment thereofalong with an anti-cancer agent can be formulated into a singlecomposition for simultaneous delivery or formulated separately into twoor more compositions (e.g., a kit). Each component can be administeredto a subject at a different time than when the other component isadministered; for example, each administration may be givennon-simultaneously (e.g., separately or sequentially) at severalintervals over a given period of time. Moreover, the separate componentsmay be administered to a subject by the same or by a different route.

In particular embodiments, the antibodies or antigen-binding fragmentsthereof disclosed herein may be used alone, or in association withother, further therapeutic agents and/or therapeutic procedures, fortreating or preventing any disease such as cancer, e.g., as discussedherein, in a subject in need of such treatment or prevention.Compositions, e.g., pharmaceutical compositions comprising apharmaceutically acceptable carrier, comprising such antibodies andfragments in association with further therapeutic agents are also partof the present invention.

Therefore, the present invention provides a method of treating cancer ina human subject, comprising administering to the subject an effectiveamount of the antibody or antigen binding fragment disclosed herein,optionally in association with a further therapeutic agent ortherapeutic procedure. The present invention also provides a method oftreating an infection or infectious disease in a human subject,comprising administering to the subject an effective amount of theantibody or antigen binding fragment disclosed herein, optionally inassociation with a further therapeutic agent or therapeutic procedure.The present invention also provides a method of increasing the activityof an immune cell, comprising administering to a subject in need thereofan effective amount of an antibody or antigen binding fragment disclosedherein. In one embodiment, the method is used for: the treatment ofcancer; the treatment of an infection or infectious disease; or as avaccine adjuvant.

In particular embodiments, the antibodies or antigen-binding fragmentsthereof disclosed herein may be used alone, or in association with tumorvaccines. Examples of tumor vaccines include but are not limited tovaccines for Human Papillomavirus (HPV) infection caused cancer such asGardasil®, Gardisil9® and Cervarix®; vaccines that prevent hepatitis Bvirus caused liver cancer such as Engerix-B® and Recombivax HB®;oncolytic virus therapy that triggers immune response such as Imlygic®;DNA vaccines such as Synchotrope MA2M plasmid DNA vaccine and ZYC101;mammaglobin-a DNA vaccine (see Clinical Cancer Res. 201420(23):5964-75); vector based vaccines such as PSA-TRICOM (prostvac),PANVAC-VF, Listeria monocytogenes-based vaccines (see, e.g., TherapeuticAdvances in Vaccines, 2014, 2(5) 137-148), Listeria-based vaccines(Listeria expressing one or more cancer vaccines such asListeria-mesothelin (e.g., CRS-207), ADXS-HPV, Axalimogene Filolisbac,Listeria-HER2/Neu, Listeria-EGFRvIII), Adeno-CEA; allogeneic vaccinessuch as GVAX, BLP-25 (anti-Ankara-mucin 1), Belagenpumatucel-L, TG4010,CIMAvax epidermal growth factor vaccine, NY-ESO, GM.CD40L-CCL21;autologous vaccines such as:Adeno-CD40L, BCG, INGN-225, Dendritic cellvaccines such as Provenge(Sipuleucel-T), rF-CEA-MUC1-TRICOM (panvac-DC);antigen vaccines such as MUC-1 (stimuvax), NY-ESO-1, GP-100, MAGE-A3(melanoma antigen encoding gene A3), INGN-225 (see Pharmacology &Therapeutics 153 (2015) 1-9).

Eat-me signals could be elevated by cytotoxic therapies likeradiotherapy or chemotherapeutic agents including, but not limited toanthracyclines (doxorubicin, epirubicin, daunorubicin, idarubicin,mitoxantrone), oxaliplatin, bortezomib, cyclophosphamide, bleomycin,vorinostat, paclitaxel, 5-fluorouracil, cytarabine, prednisolone,docetaxel, mitomycin C, topotecan/camptothecin, etoposide, zoledronicacid, methotrexate, ibrutinib, aflibercept, bevacizumab, toremifene,vinblastine, vincristine, idelalisib, mercaptopurine, thalidomide,sorafenib. Thus, in certain embodiments, the antibodies orantigen-binding fragments thereof disclosed herein may be used inassociation with chemotherapeutic agents, in association with radiationtherapy, etc. In particular embodiments, the antibodies orantigen-binding fragments thereof disclosed herein may be used alone, orin association with targeted therapies. Examples of targeted therapiesinclude: hormone therapies, signal transduction inhibitors (e.g., EGFRinhibitors, such as cetuximab (Erbitux) and erlotinib (Tarceva)); CD20inhibitors (e.g., rituximab (Rituxan) and ofatumumab (Arzerra)); CD38inhibitors (e.g., daratumumab (DARZALEX)); CD52 inhibitors (e.g.,alemtuzumab (Campath)); HER2 inhibitors (e.g., trastuzumab (Herceptin)and pertuzumab (Perjeta)); BCR-ABL inhibitors (such as imatinib(Gleevec) and dasatinib (Sprycel)); ALK inhibitors (such as crizotinib(Xalkori) and ceritinib (Zykadia)); BRAF inhibitors (such as vemurafenib(Zelboraf) and dabrafenib (Tafinlar)), gene expression modulators (e.g.,decitabine (Dacogen) and Vorinostat (Zolinza)), apoptosis inducers(e.g., bortezomib (Velcade) and carfilzomib (Kyprolis)), angiogenesisinhibitors (e.g., bevacizumab (Avastin) and ramucirumab (Cyramza)),immunomodulatory imide drugs (e.g., thalidomide, lenalidomide,pomalidomide, and apremilast), monoclonal antibodies attached to toxins(e.g., brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine(Kadcyla)).

The antibodies or antigen-binding fragments thereof disclosed herein maypreferably find use in association with targeted therapies in whichantibodies are employed to mediate ADCC/ADCP. Functional bioassays areavailable to analyze the mode of action of an antibody drug and todistinguish ADCP as a mode of action from ADCC. By way of example, anantibody-dependent cell-mediated cytotoxicity (ADCC) assay typicallyutilizes normal human peripheral blood mononuclear cells (PBMCs) oreffector cells isolated thereof. Assay variation can be reduced by usingselective donor pools with defined Fcγ receptor IIa (FcγRIIa/CD32a),IIIa (FcγRIIIa/CD16a) or IIIb (FcγRIIIb/CD16b) gene copy numbervariation (CNV) or genotypes such as FcγRIIIa-158 V/V versus V/F or F/F,FcγRIIIa-131 H/H versus H/R or R/R, and the FcγRIIIb-NA1 and -NA2polymorphic variants. Alternatively, effector cells such as PBMCs,PBMC-derived natural killer (NK) cells, granulocytes, monocytes,monocyte-derived macrophages, or dendritic cells (DCs) can be replacedwith a FcγRIIIa-expressing cell line (for example, engineered NK92).Killing of the target cells can be assessed by measuring the release ofspecific probes from pre-labelled target cells, using ⁵¹chromium (Cr⁵¹)or fluorescent dyes such as calcein-acetoxymethyl (calcein-AM),carboxyfluorescein succinimidyl ester (CFSE),2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF),europium (Eu) or propidium iodide (PI), or by measuring the release ofcytosolic enzymes such as lactate dehydrogenase (LDH) or the release ofnucleoside triphosphate (ATP).

In contrast, antibody-dependent cellular phagocytosis (ADCP) may beassessed by measuring the destruction of target cells via granulocyte,monocyte, dendritic cell, or macrophage-mediated phagocytosis. ADCPassays use PBMC-derived cells or myeloid cell lines such as HL-60,THP-1, and U937 cells differentiated into macrophages or granulocytes.Stimuli that are commonly used to induce macrophage differentiation inmonocytic cell lines include phorbol-12-myristate-13-acetate (PMA),1,25-dihydroxyvitamin D3 (VD3), and retinoic acid (RA). RA is also knownto induce terminal granulocytic differentiation of for example HL-60cells. Phagocytosis of the target cells can be assessed by monitoringeffector cells for the internalization of specific probes from targetcells pre-labelled with fluorescent dyes such as cell proliferation dyeeFluor450, CFSE, and pH-sensitive dyes including pHrodo and CypHer5E.Phagocytosis is measured by an increase in fluorescently labelledeffector cells using flow cytometry or fluorescence microscopy.“Reporter gene” assays are also available to assess ADCP. In order tomeasure ADCP function in a reporter gene assay, target cells are firstincubated with a titration of an antibody of interest. Once the antibodyis bound to its cognate target on the target cell surface, engineeredJurkat effector cells are added. If ADCP pathway activation ensues, theJurkat cells produce a luciferase product by expression of the reportergene NFAT-RE-luc2. Luciferase activity is then measured following a 4-24hour induction period, after addition of the luciferase assay reagent.The dose-dependent response in the microtiter plate-based assay can beused to quantify the relative biological activity of the therapeuticantibody compared to the dose-response curve of a suitable referenceitem.

In particular embodiments, the anti-SIRPα antibodies or antigen-bindingfragments thereof of the invention may be used in combination with ananti-cancer therapeutic agent or immunomodulatory drug such as animmunomodulatory receptor inhibitor, e.g., an antibody orantigen-binding fragment thereof that specifically binds to thereceptor.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withone or more of:

-   -   an agonist (e.g., an agonistic antibody or antigen-binding        fragment thereof, or a soluble fusion) of a TNF receptor        protein, an Immunoglobulin-like protein, a cytokine receptor, an        integrin, a signaling lymphocytic activation molecules (SLAM        proteins), an activating NK cell receptor, a Toll like receptor,        OX40, CD2, CD7, CD27, CD28, CD30, CD40, ICAM-1, LFA-1 (CD1        1a/CD18), 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR,        LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44,        NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R        gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6,        VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, ITGAM, CD1 1b,        ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C,        TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84,        CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55),        PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM        (SLAMF1, CD150, IPO-3), SLAM7, BLAME (SLAMF8), SELPLG (CD162),        LTBR, LAT, GADS, PAG/Cbp, CD19a, and a ligand that specifically        binds with CD83; or    -   an inhibitor of CD47, PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAGS,        CEACAM (e.g., CEACAM-1, -3 and/or -5), VISTA, BTLA, TIGIT,        LAIR1, IDO, TDO, CD160 and/or TGFR beta.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withone or more cyclic dinculeotides or other STING pathway agonists. STING(stimulator of interferon genes, also known as TMEM173, MITA, ERIS, andMPYS) is a transmembrane protein localized to the ER that undergoes aconformational change in response to direct binding of cyclicdinucleotides (CDNs), resulting in a downstream signaling cascadeinvolving TBK1 activation, IRF-3 phosphorylation, and production ofIFN-β and other cytokines. The STING pathway in tumor-resident hostantigen presenting c3ellss is involved in the induction of a spontaneousCD8+ T cell response against tumor-derived antigens. Activation of thispathway and the subsequent production of IFN-β also reportedlycontributes to the anti-tumor effect of radiation. STING agoinists andtheir uses are described in, for example, US20060040887, US20080286296,US20120041057, US20140205653, W02014179335, WO 2014179760,US20150056224, WO 2015185565, WO 2016096174, WO 2016145102, WO2017011444, WO 2017027645, WO 2017027646, WO 2017123657, WO 2017123669,WO 2017175147, WO 2017175156, WO 2018045204, WO 2018009648, WO2018006652, WO 2018013887, WO 2018013908, US20180002369, US20180092937,and US20180093964.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withone or more of: anti-CD47 antibody, anti-PD-1 antibody (e.g., nivolumab,pembrolizumab, anti-PDL1 antibody, anti-TIGIT antibody, anti-APRILantibody, anti-CTLA4 antibody, anti-CS1 antibody (e.g., elotuzumab),anti-KIR2DL1/2/3 antibody (e.g., lirilumab), anti-CD137 antibody (e.g.,urelumab), anti-GITR antibody (e.g., TRX518), anti-PD-L1 antibody (e.g.,BMS-936559, MSB0010718C or MPDL3280A), anti-PD-L2 antibody, anti-ILT1antibody, anti-ILT2 antibody, anti-ILT3 antibody, anti-ILT4 antibody,anti-ILT5 antibody, anti-ILT6 antibody, anti-ILT7 antibody, anti-ILT8antibody, anti-CD40 antibody, anti-OX40 antibody, anti-ICOS,anti-KIR2DL1 antibody, anti-KIR2DL2/3 antibody, anti-KIR2DL4 antibody,anti-KIR2DL5A antibody, anti-KIR2DL5B antibody, anti-KIR3DL1 antibody,anti-KIR3DL2 antibody, anti-KIR3DL3 antibody, anti-NKG2A antibody,anti-NKG2C antibody, anti-NKG2E antibody, anti-4-1BB antibody (e.g.,PF-05082566), anti-TSLP antibody, anti-IL-10 antibody, IL-10 orPEGylated IL-10, or any small organic molecule inhibitor of suchtargets.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-CD20 antibody (e.g., rituximab, ofatumumab, ocrelizumab,obinutuzumab, ocaratuzumab, ublituximab, veltuzumab, ibritumomabtiuxetan, tositumomab, BVX-20, SCT-400 or PRO131921).

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-CD38 antibody (e.g., daratumumab, isatuximab or MOR202).

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-EGFR antibody (e.g., cetuximab, CetuGEX, panitumumab,nimotuzumab, depatuxizumab or AFM-21).

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-HER2 antibody (e.g., trastuzumab, TrasGEX, pertuzumab,margetuximab or ADCT-502).

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-HER3 antibody (e.g., lumretuzumab, patritumab or LJM716).

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-CD19 antibody (e.g., inebilizumab, blinatumomab, DI-B4,MDX-1342, MEDI-551, MOR208 or 4-G7SDIE).

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-CD52 antibody (e.g., alemtuzumab).

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-EpCAM antibody (e.g., adecatumumab, catumaxomab, edrecolomab orING-1).

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-SLAMF7 antibody (e.g., elotuzumab or ABBV-838).

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-PD-1 antibody (e.g., nivolumab or pembrolizumab).

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-PD-L1 antibody (e.g., BMS-936559, MSB0010718C or MPDL3280A).

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-CTLA4 antibody (e.g., ipilimumab or tremelimumab).

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-CD137 antibody (e.g., urelumab).

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-GITR antibody (e.g., TRX518 or FPA154).

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-OX40 antibody (e.g., MEDI6469, MOXR0916 or INCAGN1949).

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-CD40 antibody (e.g., lucatumumab, dacetuzmumab, APX005M,ChiLob7/4, CP-870,893 or JNJ-64457107)In an embodiment of the invention,an anti-SIRPα antibody or antigen-binding fragment thereof of theinvention is in association with an anti-CS1 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-KIR2DL1/2/3 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-CD137 (e.g., urelumab) antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-GITR (e.g., TRX518) antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-PD-L2 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-ITL1 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-ITL2 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-ITL3 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-ITL4 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-ITL5 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-ITL6 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-ITL7 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-ITL8 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-CD40 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-OX40 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-KIR2DL1 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-KIR2DL2/3 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-KIR2DL4 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-KIR2DL5A antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-KIR2DL5B antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-KIR3DL1 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-KIR3DL2 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-KIR3DL3 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-NKG2A antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-NKG2C antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-ICOS antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-4-1BB antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-IL-10 antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withan anti-TSLP antibody.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withIL-10 or PEGylated IL-10.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withone or more of an inhibitor (e.g., a small organic molecule or anantibody or antigen-binding fragment thereof) such as: an MTOR(mammalian target of rapamycin) inhibitor, a cytotoxic agent, a platinumagent, an EGFR inhibitor, a VEGF inhibitor, a microtubule stabilizer, ataxane, a CD20 inhibitor, a CD52 inhibitor, a CD30 inhibitor, a RANK(Receptor activator of nuclear factor kappa-B) inhibitor, a RANKL(Receptor activator of nuclear factor kappa-B ligand) inhibitor, an ERKinhibitor, a MAP Kinase inhibitor, an AKT inhibitor, a MEK inhibitor, aPI3K inhibitor, a HER1 inhibitor, a HER2 inhibitor, a HERS inhibitor, aHER4 inhibitor, a Bcl2 inhibitor, a CD22 inhibitor, a CD79b inhibitor,an ErbB2 inhibitor, or a farnesyl protein transferase inhibitor.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withany one or more of: 13-cis-retinoic acid,3-[5-(methylsulfonylpiperadinemethyl)-indolyl]-quinolone,4-hydroxytamoxifen, 5-deooxyuridine, 5′-deoxy-5-fluorouridine,5-fluorouracil, 6-mecaptopurine, 7-hydroxystaurosporine, A-443654,abirateroneacetate, abraxane, ABT-578, acolbifene, ADS-100380, ALT-110,altretamine, amifostine, aminoglutethimide, amrubicin, Amsacrine,anagrelide, anastrozole, angiostatin, AP-23573, ARQ-197, arzoxifene,AS-252424, AS-605240, asparaginase, AT-9263, atrasentan, axitinib,AZD1152, Bacillus Calmette-Guerin (BCG) vaccine, batabulin, BC-210,besodutox, bevacizumab, bicalutamide, Bio111, BIO140, bleomycin,BMS-214662, BMS-247550, BMS-275291, BMS-310705, bortezomib, buserelin,busulfan, calcitriol, camptothecin, canertinib, capecitabine,carboplatin, carmustine, CC8490, Cediranib, CG-1521, CG-781,chlamydocin, chlorambucil, chlorotoxin, cilengitide, cimitidine,cisplatin, cladribine, clodronate, COL-3, CP-724714, cyclophosphamide,cyproterone, cyproteroneacetate, cytarabine, cytosinearabinoside,dacarbazine, dacinostat, dactinomycin, dalotuzumab, danusertib,dasatanib, daunorubicin, decatanib, deguelin, denileukin,deoxycoformycin, depsipeptide, diarylpropionitrile, diethylstilbestrol,diftitox, docetaxel, dovitinib, doxorubicin, droloxifene, edotecarin,yttrium-90 labeled-edotreotide, edotreotide, EKB-569, EMD121974,endostatin, enzalutamide, enzastaurin, epirubicin, epithilone B,ERA-923, Erbitux, erlotinib, estradiol, estramustine, etoposide,everolimus, exemestane, ficlatuzumab, finasteride, flavopiridol,floxuridine, fludarabine, fludrocortisone, fluoxymesterone, flutamide,FOLFOX regimen, Fulvestrant, galeterone, gefitinib, gemcitabine,gimatecan, goserelin, goserelin acetate, gossypol, GSK461364, GSK690693,HMR-3339, hydroxyprogesteronecaproate, hydroxyurea, IC87114, idarubicin,idoxyfene, ifosfamide, IM862, imatinib, IMC-1C11, INCB24360, INO1001,interferon, interleukin-12, ipilimumab, irinotecan, JNJ-16241199,ketoconazole, KRX-0402, thalidomide, lenalidomide, pomalidomide,apremilast, lapatinib, lasofoxifene, letrozole, leucovorin, leuprolide,leuprolide acetate, levamisole, liposome entrapped paclitaxel,lomustine, lonafarnib, lucanthone, LY292223, LY292696, LY293646,LY293684, LY294002, LY317615, marimastat, mechlorethamine,medroxyprogesteroneacetate, megestrolacetate, melphalan, mercaptopurine,mesna, methotrexate, mithramycin, mitomycin, mitotane, mitoxantrone,tozasertib, MLN8054, neovastat, Neratinib , neuradiab, nilotinib,nilutimide, nolatrexed, NVP-BEZ235, oblimersen, octreotide, ofatumumab,oregovomab, orteronel, oxaliplatin, paclitaxel, palbociclib,pamidronate, panitumumab, pazopanib, PD0325901, PD184352,PEG-interferon, pemetrexed, pentostatin, perifosine,phenylalaninemustard, PI-103, pictilisib, PIK-75, pipendoxifene,PKI-166, plicamycin, porfimer, prednisone, procarbazine, progestins,PX-866, R-763, raloxifene, raltitrexed, razoxin, ridaforolimus,rituximab, romidepsin, RTA744, rubitecan, scriptaid, Sdx102, seliciclib,selumetinib, semaxanib, SF1126, sirolimus, SN36093, sorafenib,spironolactone, squalamine, SR13668, streptozocin, SU6668,suberoylanalide hydroxamic acid, sunitinib, synthetic estrogen,talampanel, talimogene laherparepvec, tamoxifen, temozolomide,temsirolimus, teniposide, tesmilifene, testosterone, tetrandrine,TGX-221, thalidomide, thioguanine, thiotepa, tremelimumab, tipifarnib,tivozanib, TKI-258, TLK286, topotecan, toremifene citrate, trabectedin,trastuzumab, tretinoin, trichostatin A, triciribinephosphatemonohydrate, triptorelin pamoate, TSE-424, uracil mustard, valproicacid, valrubicin, vandetanib, vatalanib, VEGF trap, vinblastine,vincristine, vindesine, vinorelbine, vitaxin, vitespan, vorinostat,VX-745, wortmannin, Xr311, zanolimumab, ZK186619, ZK-304709, ZM336372,ZSTK474.

Non-limiting examples of suitable anti-cancer agents to be used incombination with an anti-SIRPα antibody or antigen-binding fragmentthereof of the invention include cytostatic agents, immune modulatingimide drugs, cytotoxic agents, targeted therapeutic agents (smallmolecules, biologics, siRNA and microRNA) against cancer and neoplasticdiseases,

-   -   1) anti-metabolites (such as methotrexate, 5-fluorouracil,        gemcitabine, fludarabine, capecitabine);    -   2) alkylating agents, such as temozolomide, cyclophosphamide,    -   3) DNA interactive and DNA damaging agents, such as cisplatin,        oxaliplatin, doxorubicin,    -   4) Ionizing irradiation, such as radiation therapy,    -   5) topoisomerase II inhibitors, such as etoposide, doxorubicin,    -   6) topoisomerase I inhibitors, such as irinotecan, topotecan,    -   7) tubulin interacting agents, such as paclitaxel, docetaxel,        Abraxane, epothilones,    -   8) kinesin spindle protein inhibitors,    -   9) spindle checkpoint inhibitors,    -   10) Poly(ADP-ribose) polymerase (PARP) inhibitors, such as        olaparib, MK-4827 and veliparib    -   11) Matrix metalloprotease (MMP) inhibitors    -   12) Protease inhibitors, such as cathepsin D and cathepsin K        inhibitors    -   13) Proteosome or ubiquitination inhibitors, such as bortezomib,    -   14) Activator of mutant p53 to restore its wild-type p53        activity    -   15) Adenoviral-p53    -   16) Bcl-2 inhibitors, such as ABT-263    -   17) Heat shock protein (HSP) modulators, such as geldanamycin        and 17-AAG    -   18) Histone deacetylase (HDAC) inhibitors, such as vorinostat        (SAHA),    -   19) sex hormone modulating agents,        -   a. anti-estrogens, such as tamoxifen, fulvestrant,        -   b. selective estrogen receptor modulators (SERM), such as            raloxifene,        -   c. anti-androgens, such as bicalutamide, flutamide        -   d. LHRH agonists, such as leuprolide,        -   e. 5α-reductase inhibitors, such as finasteride,        -   f. Cytochrome P450 C17 lyase (CYP450c17, also called 17αC);        -   g. aromatase inhibitors, such as letrozole, anastrozole,            exemestane,    -   20) EGFR kinase inhibitors, such as geftinib, erlotinib,        laptinib    -   21) dual erbB1 and erbB2 inhibitors, such as Lapatinib    -   22) multi-targeted kinases (serine/threonine and/or tyrosine        kinase) inhibitors,        -   a. ABL kinase inhibitors, imatinib and nilotinib, dasatinib        -   b. VEGFR-1, VEGFR-2, PDGFR, KDR, FLT, c-Kit, Tie2, Raf, MEK            and ERK inhibitors, such as sunitinib, sorafenib,            Vandetanib, pazopanib, PLX-4032, Axitinib, PTK787,            GSK-1120212        -   c. Polo-like kinase inhibitors        -   d. Aurora kinase inhibitors        -   e. JAK inhibitor        -   f. c-MET kinase inhibitors        -   g. Cyclin-dependent kinase inhibitors, such as CDK1 and CDK2            inhibitor Dinaciclib SCH 727965 (see Parry et al, Molecular            Cancer Therapeutics 9 (8): 2344-53 (2010)) and CDK4/6            inhibitors, such as Ribociclib, Palbociclib, Abemaciclib,            and Trilaciclib.        -   h. PI3K and mTOR inhibitors, such as GDC-0941, BEZ-235,            BKM-120 and AZD-8055        -   i. Rapamycin and its analogs, such as Temsirolimus,            everolimus, and deforolimus    -   23) and other anti-cancer (also know as anti-neoplastic) agents        include but are not limited to ara-C, adriamycin, cytoxan,        Carboplatin, Uracil mustard, Clormethine, Ifosfsmide, Melphalan,        Chlorambucil, Pipobroman, Triethylenemelamine,        Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,        Streptozocin, Dacarbazine, Floxuridine, Cytarabine,        6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate,        Pentostatine, Vinblastine, Vincristine, Vindesine, Vinorelbine,        Navelbine, Bleomycin, Dactinomycin, Daunorubicin, Doxorubicin,        Epirubicin, teniposide, cytarabine, pemetrexed, Idarubicin,        Mithramycin, Deoxycoformycin, Mitomycin-C, L-Asparaginase,        Teniposide, Ethinylestradiol, Diethylstilbestrol, Testosterone,        Prednisone, Fluoxymesterone, Dromostanolone propionate,        Testolactone, Megestrolacetate, Methylprednisolone,        Methyltestosterone, Prednisolone, Triamcinolone,        Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide,        Estramustine, Flutamide Medroxyprogesteroneacetate, Toremifene,        goserelin, Carboplatin, Hydroxyurea, Amsacrine, Procarbazine,        Mitotane, Mitoxantrone, Levamisole, Drolloxafine,        Hexamethylmelamine, Bexxar, Zevalin, Trisenox, Profimer,        Thiotepa, Altretamine, Doxil, Ontak, Depocyt, Aranesp, Neupogen,        Neulasta, Kepivance.    -   24) Farnesyl protein transferase inhibitors, such as,        SARASAR™(4-[2-[4-[(11R)-3,10-dibromo-8-chloro-6,        11-dihydro-5H-benzo        [5,6]cyclohepta[1,2-b]pyridin-11-yl-]-1-piperidinyl]-2-oxoethyl]-piperidinecarboxamide,        tipifarnib    -   25) interferons, such as Intron A, Peg-Intron,    -   26) anti-erbB1 antibodies, such as cetuximab, panitumumab,    -   27) anti-erbB2 antibodies, such as trastuzumab,    -   28) anti-CD52 antibodies, such as Alemtuzumab,    -   29) anti-CD20 antibodies, such as Rituximab    -   30) anti-CD33 antibodies, such as Gemtuzumab ozogamicin    -   31) anti-VEGF antibodies, such as Avastin,    -   32) TRIAL ligands, such as Lexatumumab, mapatumumab, and AMG-655    -   33) anti-CTLA-4 antibodies, such as ipilimumab    -   34) antibodies against CTA1, CEA, CDS, CD19, CD22, CD30, CD44,        CD44V6, CD55, CD56, EpCAM, FAP, MHCII, HGF, IL-6, MUC1, PSMA,        TAL6, TAG-72, TRAILR, VEGFR, IGF-2, FGF,    -   35) anti-IGF-1R antibodies, such as dalotuzumab (MK-0646) and        robatumumab (SCH 717454).

“Estrogen receptor modulators” refers to compounds that interfere withor inhibit the binding of estrogen to the receptor, regardless ofmechanism. Examples of estrogen receptor modulators include, but are notlimited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081,toremifene, fulvestrant,4-[7-(2,2-dimethyl-l-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

“Androgen receptor modulators” refers to compounds which interfere orinhibit the binding of androgens to the receptor, regardless ofmechanism. Examples of androgen receptor modulators include finasterideand other 5a-reductase inhibitors, nilutamide, flutamide, bicalutamide,liarozole, and abiraterone acetate.

“Retinoid receptor modulators” refers to compounds which interfere orinhibit the binding of retinoids to the receptor, regardless ofmechanism. Examples of such retinoid receptor modulators includebexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid,α-difluoromethylornithine, ILX23-7553, trans-N-(4′ -hydroxyphenyl)retinamide, and N-4-carboxyphenyl retinamide.

“Cytotoxic/cytostatic agents” refer to compounds which cause cell deathor inhibit cell proliferation primarily by interfering directly with thecell's functioning or inhibit or interfere with cell myosis, includingalkylating agents, tumor necrosis factors, intercalators, hypoxiaactivatable compounds, microtubule inhibitors/microtubule-stabilizingagents, inhibitors of mitotic kinesins, histone deacetylase inhibitors,inhibitors of kinases involved in mitotic progression, inhibitors ofkinases involved in growth factor and cytokine signal transductionpathways, antimetabolites, biological response modifiers,hormonal/anti-hormonal therapeutic agents, haematopoietic growthfactors, monoclonal antibody targeted therapeutic agents, topoisomeraseinhibitors, proteosome inhibitors, ubiquitin ligase inhibitors, andaurora kinase inhibitors.

Examples of cytotoxic/cytostatic agents include, but are not limited to,platinum coordinator compounds, sertenef, cachectin, ifosfamide,tasonermin, lonidamine, carboplatin, altretamine, prednimustine,dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin,temozolomide, heptaplatin, estramustine, improsulfan tosilate,trofosfamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin,satraplatin, profiromycin, cisplatin, irofulven, dexifosfamide,cis-aminedichloro(2-methyl-pyridine)platinum, benzylguanine,glufosfamide, GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum (II)]tetrachloride, diarizidinylspermine,arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin,pinafide, valrubicin, amrubicin, antineoplaston,3′-deamino-3′-morpholino-13-deoxo-10-hydroxycarminomycin, annamycin,galarubicin, elinafide, MEN10755,4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin (seeWO 00/50032).

An example of a hypoxia activatable compound is tirapazamine.

Examples of proteosome inhibitors include but are not limited tolactacystin and MLN-341 (Velcade).

Examples of microtubule inhibitors/microtubule-stabilising agentsinclude taxanes in general. Specific compounds include paclitaxel(Taxol®), vindesine sulfate,3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxol(Taxotere®), rhizoxin, dolastatin, mivobulin isethionate, auristatin,cemadotin, RPR109881, BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl) benzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide,TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and6,288,237) and BMS188797.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine,irinotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)propanamine,1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione,lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350,BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane,2′-dimethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide,asulacrine, (5a, 5aB,8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydro0xy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxo1-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium,6,9-bis[(2-aminoethyl)amino]benzo[g]isoguinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one,N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide,N-(2-(dimethylamino)ethyl)acridine-4-carboxamide,6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one, and dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the humanmitotic kinesin KSP, are described in Publications WO03/039460,WO03/050064, WO03/050122, WO03/049527, WO03/049679, WO03/049678,WO04/039774, WO03/079973, WO03/099211, WO03/105855, WO03/106417,WO04/037171, WO04/058148, WO04/058700, WO04/126699, WO05/018638,WO05/019206, WO05/019205, WO05/018547, WO05/017190, US2005/0176776. Inan embodiment inhibitors of mitotic kinesins include, but are notlimited to inhibitors of KSP, inhibitors of MKLP1, inhibitors of CENP-E,inhibitors of MCAK and inhibitors of Rab6-KIFL.

Examples of “histone deacetylase inhibitors” include, but are notlimited to, SAHA, TSA, oxamflatin, PXD101, MG98 and scriptaid. Furtherreference to other histone deacetylase inhibitors may be found in thefollowing manuscript; Miller, T. A. et al. J. Med. Chem.46(24):5097-5116 (2003).

“Inhibitors of kinases involved in mitotic progression” include, but arenot limited to, inhibitors of aurora kinase, inhibitors of Polo-likekinases (PLK; in particular inhibitors of PLK-1), inhibitors of bub-1and inhibitors of bub-R1. An example of an “aurora kinase inhibitor” isVX-680.

“Antiproliferative agents” includes antisense RNA and DNAoligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001,and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin,doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine,cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed,paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed,nelzarabine, 2′-deoxy-2′-methylidenecytidine,2′-fluoromethylene-2′-deoxycytidine,N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea,N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine,aplidine, ecteinascidin, troxacitabine,4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamicacid, aminopterin, 5-flurouracil, alanosine,11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-ylacetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N4-palmitoyl-1-B-D-arabino furanosyl cytosine,3-aminopyridine-2-carboxaldehyde thiosemicarbazone and trastuzumab.

Examples of monoclonal antibody targeted therapeutic agents includethose therapeutic agents which have cytotoxic agents or radioisotopesattached to a cancer cell specific or target cell specific monoclonalantibody. Examples include Bexxar.

“Prenyl-protein transferase inhibitor” refers to a compound whichinhibits any one or any combination of the prenyl-protein transferaseenzymes, including farnesyl-protein transferase (FPTase),geranylgeranyl-protein transferase type I (GGPTase-I), andgeranylgeranyl-protein transferase type-II (GGPTase-II, also called RabGGPTase).

Examples of prenyl-protein transferase inhibitors can be found in thefollowing publications and patents: WO 96/30343, WO 97/18813, WO97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO95/32987, U.S. Pat. Nos. 5,420,245, 5,523,430, 5,532,359, 5,510,510,5,589,485, 5,602,098, European Patent Publ. 0 618 221, European PatentPubl. 0 675 112, European Patent Publ. 0 604 181, European Patent Publ.0 696 593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO 95/10515, WO95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO 96/33159, WO96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO98/02436, and U.S. Pat. No. 5,532,359. For an example of the role of aprenyl-protein transferase inhibitor on angiogenesis see European J. ofCancer, Vol. 35, No. 9, pp.1394-1401 (1999).

“Angiogenesis inhibitors” refers to compounds that inhibit the formationof new blood vessels, regardless of mechanism. Examples of angiogenesisinhibitors include, but are not limited to, tyrosine kinase inhibitors,such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) andFlk-1/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived,or platelet derived growth factors, MMP (matrix metalloprotease)inhibitors, integrin blockers, interferon-a, interleukin-12, pentosanpolysulfate, cyclooxygenase inhibitors, including nonsteroidalanti-inflammatories (NSAIDs) like aspirin and ibuprofen as well asselective cyclooxy-genase-2 inhibitors like celecoxib and rofecoxib(PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch.Opthalmol., Vol. 108, p.573 (1990); Anat. Rec., Vol. 238, p. 68 (1994);FEBS Letters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol. 313, p. 76(1995); J. Mol. Endocrinol., Vol. 16, p. 107 (1996); Jpn. J. Pharmacol.,Vol. 75, p. 105 (1997); Cancer Res., Vol. 57, p. 1625 (1997); Cell, Vol.93, p. 705 (1998); Intl. J. Mol. Med., Vol. 2, p. 715 (1998); J. Biol.Chem., Vol. 274, p. 9116 (1999)), steroidal anti-inflammatories (such ascorticosteroids, mineralocorticoids, dexamethasone, prednisone,prednisolone, methylpred, betamethasone), carboxyamidotriazole,combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol,thalidomide, angiostatin, troponin-1, angiotensin II antagonists (seeFernandez et al., J. Lab. Clin. Med. 105: 141-145 (1985)), andantibodies to VEGF (see, Nature Biotechnology, Vol. 17, pp. 963-968(October 1999); Kim et al., Nature, 362, 841-844 (1993); WO 00/44777;and WO 00/61186).

Other examples of angiogenesis inhibitors include, but are not limitedto, endostatin, ukrain, ranpirnase, IM862,5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate,acetyldinanaline,5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide,CM101, squalamine, combretastatin, RPI4610, NX31838, sulfatedmannopentaose phosphate,7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalenedisulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone(SU5416).

Other therapeutic agents that modulate or inhibit angiogenesis and mayalso be used in combination with the compounds of the instant inventioninclude agents that modulate or inhibit the coagulation and fibrinolysissystems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examplesof such agents that modulate or inhibit the coagulation and fibrinolysispathways include, but are not limited to, heparin (see Thromb. Haemost.80:10-23 (1998)), low molecular weight heparins and carboxypeptidase Uinhibitors (also known as inhibitors of active thrombin activatablefibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354(2001)). TAFIa inhibitors have been described in U.S. Ser. Nos.60/310,927 (filed Aug. 8, 2001) and 60/349,925 (filed Jan. 18, 2002).

“Agents that interfere with cell cycle checkpoints” refer to compoundsthat inhibit protein kinases that transduce cell cycle checkpointsignals, thereby sensitizing the cancer cell to DNA damaging agents.Such agents include inhibitors of ATR, ATM, the CHK11 and CHK12 kinasesand cdk and cdc kinase inhibitors and are specifically exemplified by7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

“Agents that interfere with receptor tyrosine kinases (RTKs)” refer tocompounds that inhibit RTKs and therefore mechanisms involved inoncogenesis and tumor progression. Such agents include inhibitors ofc-Kit, Eph, PDGF, Flt3 and c-Met. Further agents include inhibitors ofRTKs as described by Bume-Jensen and Hunter, Nature, 411:355-365, 2001.

“Inhibitors of cell proliferation and survival signalling pathway” referto compounds that inhibit signal transduction cascades downstream ofcell surface receptors. Such agents include inhibitors ofserine/threonine kinases (including but not limited to inhibitors of Aktsuch as described in WO 02/083064, WO 02/083139, WO 02/083140, US2004-0116432, WO 02/083138, US 2004-0102360, WO 03/086404, WO 03/086279,WO 03/086394, WO 03/084473, WO 03/086403, WO 2004/041162, WO2004/096131, WO 2004/096129, WO 2004/096135, WO 2004/096130, WO2005/100356, WO 2005/100344, US 2005/029941, US 2005/44294, US2005/43361, 60/734188, 60/652737, 60/670469), inhibitors of Raf kinase(for example PLX-4032), inhibitors of MEK (for example Arry-162,RO-4987655 and GSK-1120212), inhibitors of mTOR (for example AZD-8055,BEZ-235 and everolimus), and inhibitors of PI3K (for example GDC-0941,BKM-120).

As used above, “integrin blockers” refers to compounds which selectivelyantagonize, inhibit or counteract binding of a physiological ligand tothe α_(v)β3integrin, to compounds which selectively antagonize, inhibitor counteract binding of a physiological ligand to the avβ5 integrin, tocompounds which antagonize, inhibit or counteract binding of aphysiological ligand to both the a_(V)β₃ integrin and the α_(V)β₅integrin, and to compounds which antagonize, inhibit or counteract theactivity of the particular integrin(s) expressed on capillaryendothelial cells. The term also refers to antagonists of the α_(v)β₆,α_(v)β₈, α₂β₁, α₂β₁, α₅β₁, α₆β₁, and α₆β₄ integrins. The term alsorefers to antagonists of any combination of α_(v)β₃, α_(v)β₅, α_(v)β₈,α₁β₁, α₂β₁, α₅β₁, α₆β₁, and α₆β₄ integrins.

Some specific examples of tyrosine kinase inhibitors includeN-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline,N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine,BIBX1382,2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one,SH268, genistein, STI571, CEP2563,4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethanesulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571A,N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and EMD121974.

Combinations of the instantly claimed antibodies or antigen bindingfragments with PPAR-γ (i.e., PPAR-gamma) agonists and PPAR-δ (i.e.,PPAR-delta) agonists may be useful in the treatment of certainmalignancies. PPAR-γ and PPAR-δ are the nuclear peroxisomeproliferator-activated receptors γ and δ. The expression of PPAR-γ onendothelial cells and its involvement in angiogenesis has been reportedin the literature (see J. Cardiovasc. Pharmacol. 1998; 31: 909-913; J.Biol. Chem. 1999; 274: 9116-9121; Invest. Ophthalmol Vis. Sci. 2000; 41:2309-2317). More recently, PPAR-γ agonists have been shown to inhibitthe angiogenic response to VEGF in vitro; both troglitazone androsiglitazone maleate inhibit the development of retinalneovascularization in mice. (Arch. Ophthamol. 2001; 119: 709-717).Examples of PPAR-γ agonists and PPAR-γ/α agonists include, but are notlimited to, Lynparza®, Rucaparib®, Talazoparib®, niraparib, Veliparib®,thiazolidinediones (such as DRF2725, CS-011, troglitazone,rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate,GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544,NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926,2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionicacid, and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane-2-carboxylic acid.

The antibody or antigen binding fragment of the instant invention mayalso be useful for treating or preventing breast cancer in combinationwith aromatase inhibitors. Examples of aromatase inhibitors include butare not limited to: anastrozole, letrozole and exemestane.

The antibody or antigen binding fragment of the instant invention mayalso be useful for treating cancer in combination with the followingchemotherapeutic agents: abarelix (Plenaxis depot®); aldesleukin(Prokine®); Aldesleukin (Proleukin®); Alemtuzumab (Campath®);alitretinoin (Panretin®); allopurinol (Zyloprim®); altretamine(Hexalen®); amifostine (Ethyol®); anastrozole (Arimidex®); arsenictrioxide (Trisenox®); asparaginase (Elspar®); azacitidine (Vidaza®);bendamustine hydrochloride (Treanda®); bevacuzimab (Avastin®);bexarotene capsules (Targretin®); bexarotene gel (Targretin®); bleomycin(Blenoxane®); bortezomib (Velcade®); brefeldin A; busulfan intravenous(Busulfex®); busulfan oral (Myleran®); calusterone (Methosarb®);capecitabine (Xeloda®); carboplatin (Paraplatin®); carmustine (BCNU®,BiCNU®); carmustine (Gliadel®); carmustine with Polifeprosan 20 Implant(Gliadel Wafer®); celecoxib (Celebrex®); cetuximab (Erbitux®);chlorambucil (Leukeran®); cisplatin (Platinol®); cladribine (Leustatin®,2-CdA®); clofarabine (Clolar®); cyclophosphamide (Cytoxan®, Neosar®);cyclophosphamide (Cytoxan Injection®); cyclophosphamide (CytoxanTablet®); cytarabine (Cytosar-U®); cytarabine liposomal (DepoCyt®);dacarbazine (DTIC-Dome®); dactinomycin, actinomycin D (Cosmegen®);dalteparin sodium injection (Fragmin®); daratumumab (DARZALEX®);Darbepoetin alfa (Aranesp®); dasatinib (Sprycel®); daunorubicinliposomal (DanuoXome®); daunorubicin, daunomycin (Daunorubicin®);daunorubicin, daunomycin (Cerubidine®); degarelix (Firmagon®);Denileukin diftitox (Ontak®); dexrazoxane (Zinecard®); dexrazoxanehydrochloride (Totect®); didemnin B; 17-DMAG; docetaxel (Taxotere®);doxorubicin (Adriamycin PFS®); doxorubicin (Adriamycin®, Rubex®);doxorubicin (Adriamycin PFS Injection®); doxorubicin liposomal (Doxil®);dromostanolone propionate (Dromostanolone®); dromostanolone propionate(Masterone Injection®); eculizumab injection (Soliris®); Elliott's BSolution (Elliott's B Solution®); eltrombopag (Promacta®); epirubicin(Ellence®); Epoetin alfa (epogen®); erlotinib (Tarceva®); estramustine(Emcyt®); ethinyl estradiol; etoposide phosphate (Etopophos®);etoposide, VP-16 (Vepesid®); everolimus tablets (Afinitor®); exemestane(Aromasin®); ferumoxytol (Feraheme Injection®); Filgrastim (Neupogen®);floxuridine (intraarterial) (FUDR®); fludarabine (Fludara®);fluorouracil, 5-FU (Adrucil®); fulvestrant (Faslodex®); gefitinib(Iressa®); geldanamycin; gemcitabine (Gemzar®); gemtuzumab ozogamicin(Mylotarg®); goserelin acetate (Zoladex Implant®); goserelin acetate(Zoladex®); histrelin acetate (Histrelin implant®); hydroxyurea(Hydrea®); Ibritumomab Tiuxetan (Zevalin®); idarubicin (Idamycin®);ifosfamide (IFEX®); imatinib mesylate (Gleevec®); interferon alfa 2a(Roferon A®); Interferon alfa-2b (Intron A®); iobenguane I 123 injection(AdreView®); irinotecan (Camptosar®); ixabepilone (Ixempra®); lapatinibtablets (Tykerb®); lenalidomide (Revlimid®); letrozole (Femara®);leucovorin (Wellcovorin®, Leucovorin®); Leuprolide Acetate (Eligard®);levamisole (Ergamisol®); lomustine, CCNU (CeeBU®); meclorethamine,nitrogen mustard (Mustargen®); megestrol acetate (Megace®); melphalan,L-PAM (Alkeran®); mercaptopurine, 6-MP (Purinethol®); mesna (Mesnex®);mesna (Mesnex tabs®); methotrexate (Methotrexate®); methoxsalen(Uvadex®); 8-methoxypsoralen; mitomycin C (Mutamycin®); mitotane(Lysodren®); mitoxantrone (Novantrone®); mitramycin; nandrolonephenpropionate (Durabolin-50®); nelarabine (Arranon®); nilotinib(Tasigna®); Nofetumomab (Verluma®); ofatumumab (Arzerra®); Oprelvekin(Neumega®); oxaliplatin (Eloxatin®); paclitaxel (Paxene®); paclitaxel(Taxol®); paclitaxel protein-bound particles (Abraxane®); palifermin(Kepivance®); pamidronate (Aredia®); panitumumab (Vectibix®); pazopanibtablets (Votrienttm®); pegademase (Adagen (Pegademase Bovine)®);pegaspargase (Oncaspar®); Pegfilgrastim (Neulasta®); pemetrexed disodium(Alimta®); pentostatin (Nipent®); pipobroman (Vercyte®); plerixafor(Mozobil®); plicamycin, mithramycin (Mithracin®); porfimer sodium(Photofrin®); pralatrexate injection (Folotyn®); procarbazine(Matulane®); quinacrine (Atabrine®); rapamycin; Rasburicase (Elitek®);raloxifene hydrochloride (Evista®); Rituximab (Rituxan®); romidepsin(Istodax®); romiplostim (Nplate®); sargramostim (Leukine®); Sargramostim(Prokine®); sorafenib (Nexavar®); streptozocin (Zanosar®); sunitinibmaleate (Sutent®); talc (Sclerosol®); tamoxifen (Nolvadex®);temozolomide (Temodar®); temsirolimus (Torisel®); teniposide, VM-26(Vumon®); testolactone (Teslac®); thioguanine, 6-TG (Thioguanine®);thiopurine; thiotepa (Thioplex®); topotecan (Hycamtin®); toremifene(Fareston®); Tositumomab (Bexxar®); Tositumomab/I-131 tositumomab(Bexxar®); trans-retinoic acid; Trastuzumab (Herceptin®); tretinoin,ATRA (Vesanoid®); triethylenemelamine; Uracil Mustard (Uracil MustardCapsules®); valrubicin (Valstar®); vinblastine (Velban®); vincristine(Oncovin®); vinorelbine (Navelbine®); vorinostat (Zolinza®); wortmannin;and zoledronate (Zometa®).

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is in association withone or more antiemetics including, but not limited to: casopitant(GlaxoSmithKline), Netupitant (MGI-Helsinn) and other NK-1 receptorantagonists, palonosetron (sold as Aloxi by MGI Pharma), aprepitant(sold as Emend by Merck and Co.; Rahway, N.J.), diphenhydramine (sold asBenadryl® by Pfizer; New York, N.Y.), hydroxyzine (sold as Atarax® byPfizer; New York, N.Y.), metoclopramide (sold as Reglan® by AH RobinsCo,; Richmond, Va.), lorazepam (sold as Ativan® by Wyeth; Madison,N.J.), alprazolam (sold as Xanax® by Pfizer; New York, N.Y.),haloperidol (sold as Haldol® by Ortho-McNeil; Raritan, N.J.), droperidol(Inapsine®), dronabinol (sold as Marinol® by Solvay Pharmaceuticals,Inc.; Marietta, Ga.), dexamethasone (sold as Decadron® by Merck and Co.;Rahway, N.J.), methylprednisolone (sold as Medrol® by Pfizer; New York,N.Y.), prochlorperazine (sold as Compazine® by Glaxosmithkline; ResearchTriangle Park, N.C.), granisetron (sold as Kytril® by Hoffmann-La RocheInc.; Nutley, N.J.), ondansetron (sold as Zofran® by Glaxosmithkline;Research Triangle Park, N.C.), dolasetron (sold as Anzemet® bySanofi-Aventis; New York, N.Y.), tropisetron (sold as Navoban® byNovartis; East Hanover, N.J.).

Other side effects of cancer treatment include red and white blood celldeficiency. Accordingly, in an embodiment of the invention, ananti-SIRPα antibody or antigen-binding fragment thereof is inassociation with an agent which treats or prevents such a deficiency,such as, e.g., filgrastim, PEG-filgrastim, erythropoietin, epoetin alfaor darbepoetin alfa.

In an embodiment of the invention, an anti-SIRPα antibody orantigen-binding fragment thereof of the invention is administered inassociation with anti-cancer radiation therapy. For example, in anembodiment of the invention, the radiation therapy is external beamtherapy (EBT): a method for delivering a beam of high-energy X-rays tothe location of the tumor. The beam is generated outside the patient(e.g., by a linear accelerator) and is targeted at the tumor site. TheseX-rays can destroy the cancer cells and careful treatment planningallows the surrounding normal tissues to be spared. No radioactivesources are placed inside the patient's body. In an embodiment of theinvention, the radiation therapy is proton beam therapy: a type ofconformal therapy that bombards the diseased tissue with protons insteadof X-rays. In an embodiment of the invention, the radiation therapy isconformal external beam radiation therapy: a procedure that usesadvanced technology to tailor the radiation therapy to an individual'sbody structures. In an embodiment of the invention, the radiationtherapy is brachytherapy: the temporary placement of radioactivematerials within the body, usually employed to give an extra dose—orboost—of radiation to an area.

In an embodiment of the invention, a surgical procedure is administeredin association with an anti-SIRPα antibody or antigen-binding fragmentthereof is surgical tumorectomy.

Experimental and Diagnostic Uses

The anti-SIRPα antibodies and antigen-binding fragments thereofdisclosed herein may be used as affinity purification agents. In thisprocess, the anti-SIRPα antibodies and antigen-binding fragments thereofare immobilized on a solid phase such a Sephadex, glass or agarose resinor filter paper, using methods well known in the art. The immobilizedantibody or fragment is contacted with a sample containing the SIRPαprotein (or a fragment thereof) to be purified, and thereafter thesupport is washed with a suitable solvent that will remove substantiallyall the material in the sample except the SIRPα protein, which is boundto the immobilized antibody or fragment. Finally, the support is washedwith a solvent which elutes the bound SIRPα (e.g., protein A). Suchimmobilized antibodies and fragments form part of the present invention.

Further provided are antigens for generating secondary antibodies whichare useful for example for performing Western blots and otherimmunoassays discussed herein.

Anti-SIRPα antibodies (e.g., humanized antibodies) and antigen-bindingfragments thereof may also be useful in diagnostic assays for SIRPαprotein, e.g., detecting its expression in specific cells, tissues, orserum, e.g., myeloid cells such as monocytes, macrophages, neutrophils,basophils, eosinophils, and dendritic cells. Such diagnostic methods maybe useful in various disease diagnoses.

The present invention includes ELISA assays (enzyme-linked immunosorbentassay) incorporating the use of an anti-SIRPα antibody orantigen-binding fragment thereof disclosed herein.

For example, such a method comprises the following steps:

-   (a) coat a substrate (e.g., surface of a microtiter plate well,    e.g., a plastic plate) with anti-SIRPα antibody or antigen-binding    fragment thereof;-   (b) apply a sample to be tested for the presence of SIRPα to the    substrate;-   (c) wash the plate, so that unbound material in the sample is    removed;-   (d) apply detectably labeled antibodies (e.g., enzyme-linked    antibodies) which are also specific to the SIRPα antigen;-   (e) wash the substrate, so that the unbound, labeled antibodies are    removed;-   (f) if the labeled antibodies are enzyme linked, apply a chemical    which is converted by the enzyme into a fluorescent signal; and-   (g) detect the presence of the labeled antibody.

Detection of the label associated with the substrate indicates thepresence of the SIRPα protein.

In a further embodiment, the labeled antibody or antigen-bindingfragment thereof is labeled with peroxidase which react with ABTS (e.g.,2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) or3,3′,5,5′-Tetramethylbenzidine to produce a color change which isdetectable. Alternatively, the labeled antibody or fragment is labeledwith a detectable radioisotope (e.g., ³H) which can be detected byscintillation counter in the presence of a scintillant.

An anti-SIRPα antibody or antigen-binding fragment thereof of theinvention may be used in a Western blot or immune-protein blotprocedure. Such a procedure forms part of the present invention andincludes e.g.:

-   -   (1) optionally transferring proteins from a sample to be tested        for the presence of SIRPα (e.g., from a PAGE or SDS-PAGE        electrophoretic separation of the proteins in the sample) onto a        membrane or other solid substrate using a method known in the        art (e.g., semi-dry blotting or tank blotting); contacting the        membrane or other solid substrate to be tested for the presence        of bound SIRPα or a fragment thereof with an anti-SIRPα antibody        or antigen-binding fragment thereof of the invention.    -   (2) washing the membrane one or more times to remove unbound        anti-SIRPα antibody or fragment and other unbound substances;        and    -   (3) detecting the bound anti-SIRPα antibody or fragment.

Such a membrane may take the form of a nitrocellulose or vinyl-based(e.g., polyvinylidene fluoride (PVDF)) membrane to which the proteins tobe tested for the presence of SIRPα in a non-denaturing PAGE(polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) gel have been transferred(e.g., following electrophoretic separation in the gel). Beforecontacting the membrane with the anti-SIRPα antibody or fragment, themembrane is optionally blocked, e.g., with non-fat dry milk or the likeso as to bind non-specific protein binding sites on the membrane.

Detection of the bound antibody or fragment indicates that the SIRPαprotein is present on the membrane or substrate and in the sample.Detection of the bound antibody or fragment may be by binding theantibody or fragment with a secondary antibody (an anti-immunoglobulinantibody) which is detectably labeled and, then, detecting the presenceof the secondary antibody.

The anti-SIRPα antibodies and antigen-binding fragments thereofdisclosed herein may also be used for immunohistochemistry. Such amethod forms part of the present invention and comprises, e.g.,

-   -   (1) contacting a cell (e.g., a sample containing myeloid cells        such as monocytes, macrophages, neutrophils, basophils,        eosinophils, and dendritic cells) to be tested for the presence        of SIRPα protein with an anti-SIRPα antibody or antigen-binding        fragment thereof of the invention; and    -   (2) detecting the antibody or fragment on or in the cell.

If the antibody or fragment itself is detectably labeled, it can bedetected directly. Alternatively, the antibody or fragment may be boundby a detectably labeled secondary antibody which is detected.

Certain anti-SIRPα antibodies and antigen-binding fragments thereofdisclosed herein may also be used for in vivo tumor imaging. Such amethod may include injection of a radiolabeled anti-SIRPα antibody orantigen-binding fragment thereof into the body of a patient to be testedfor the presence of a tumor associated with SIRPα expression (e.g.,which expresses SIRPα, for example, on the tumor cell surface) followedby nuclear imaging of the body of the patient to detect the presence ofthe labeled antibody or fragment e.g., at loci comprising a highconcentration of the antibody or fragment which are bound to the tumor.The detection of the loci indicates the presence of the SIRPα⁺ tumor andtumor cells.

Imaging techniques include SPECT imaging (single photon emissioncomputed tomography) or PET imaging (positron emission tomography).Labels include e.g., iodine-123 (¹²³I) and technetium-99m (^(99m)Tc),e.g., in conjunction with SPECT imaging or ¹¹C, ¹³N, ¹⁵O or ¹⁸F, e.g.,in conjunction with PET imaging or Indium-111 (See e.g., Gordon et al.,(2005) International Rev. Neurobiol. 67:385-440).

Pharmaceutical Compositions and Administration

To prepare pharmaceutical or sterile compositions of the anti-SIRPαantibodies and antigen-binding fragments of the invention, the antibodyor antigen-binding fragment thereof is admixed with a pharmaceuticallyacceptable carrier or excipient. See, e.g., Remington's PharmaceuticalSciences and U.S. Pharmacopeia: National Formulary, Mack PublishingCompany, Easton, Pa. (1984).

Formulations of therapeutic and diagnostic agents may be prepared bymixing with acceptable carriers, excipients, or stabilizers in the formof, e.g., lyophilized powders, slurries, aqueous solutions orsuspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's ThePharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro (2000) Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.)(1993) Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.).

Toxicity and therapeutic efficacy of the antibodies of the invention,administered alone or in combination with another therapeutic agent, canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index (LD₅₀/ED₅₀). The data obtained fromthese cell culture assays and animal studies can be used in formulatinga range of dosage for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administration.

In a further embodiment, a further therapeutic agent that isadministered to a subject in association with an anti-SIRPα antibody orantigen-binding fragment thereof of the invention in accordance with thePhysicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (Nov.1, 2002)).

The mode of administration can vary. Routes of administration includeoral, rectal, transmucosal, intestinal, parenteral; intramuscular,subcutaneous, intradermal, intramedullary, intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, intraocular,inhalation, insufflation, topical, cutaneous, transdermal, orintra-arterial.

In particular embodiments, the anti-SIRPα antibodies or antigen-bindingfragments thereof of the invention can be administered by an invasiveroute such as by injection. In further embodiments of the invention, ananti-SIRPα antibody or antigen-binding fragment thereof, orpharmaceutical composition thereof, is administered intravenously,subcutaneously, intramuscularly, intraarterially, intratumorally, or byinhalation, aerosol delivery. Administration by non-invasive routes(e.g., orally; for example, in a pill, capsule or tablet) is also withinthe scope of the present invention.

The present invention provides a vessel (e.g., a plastic or glass vial,e.g., with a cap or a chromatography column, hollow bore needle or asyringe cylinder) comprising any of the antibodies or antigen-bindingfragments of the invention or a pharmaceutical composition thereof. Thepresent invention also provides an injection device comprising any ofthe antibodies or antigen-binding fragments of the invention or apharmaceutical composition thereof. An injection device is a device thatintroduces a substance into the body of a patient via a parenteralroute, e.g., intramuscular, subcutaneous or intravenous. For example, aninjection device may be a syringe (e.g., pre-filled with thepharmaceutical composition, such as an auto-injector) which, forexample, includes a cylinder or barrel for holding fluid to be injected(e.g., antibody or fragment or a pharmaceutical composition thereof), aneedle for piecing skin and/or blood vessels for injection of the fluid;and a plunger for pushing the fluid out of the cylinder and through theneedle bore. In an embodiment of the invention, an injection device thatcomprises an antibody or antigen-binding fragment thereof of the presentinvention or a pharmaceutical composition thereof is an intravenous (IV)injection device. Such a device includes the antibody or fragment or apharmaceutical composition thereof in a cannula or trocar/needle whichmay be attached to a tube which may be attached to a bag or reservoirfor holding fluid (e.g., saline; or lactated ringer solution comprisingNaCl, sodium lactate, KCl, CaCl₂ and optionally including glucose)introduced into the body of the patient through the cannula ortrocar/needle. The antibody or fragment or a pharmaceutical compositionthereof may, in an embodiment of the invention, be introduced into thedevice once the trocar and cannula are inserted into the vein of asubject and the trocar is removed from the inserted cannula. The IVdevice may, for example, be inserted into a peripheral vein (e.g., inthe hand or arm); the superior vena cava or inferior vena cava, orwithin the right atrium of the heart (e.g., a central IV); or into asubclavian, internal jugular, or a femoral vein and, for example,advanced toward the heart until it reaches the superior vena cava orright atrium (e.g., a central venous line). In an embodiment of theinvention, an injection device is an autoinjector; a jet injector or anexternal infusion pump. A jet injector uses a high-pressure narrow jetof liquid which penetrate the epidermis to introduce the antibody orfragment or a pharmaceutical composition thereof to a patient's body.External infusion pumps are medical devices that deliver the antibody orfragment or a pharmaceutical composition thereof into a patient's bodyin controlled amounts. External infusion pumps may be poweredelectrically or mechanically. Different pumps operate in different ways,for example, a syringe pump holds fluid in the reservoir of a syringe,and a moveable piston controls fluid delivery, an elastomeric pump holdsfluid in a stretchable balloon reservoir, and pressure from the elasticwalls of the balloon drives fluid delivery. In a peristaltic pump, a setof rollers pinches down on a length of flexible tubing, pushing fluidforward. In a multi-channel pump, fluids can be delivered from multiplereservoirs at multiple rates.

The pharmaceutical compositions disclosed herein may also beadministered with a needleless hypodermic injection device; such as thedevices disclosed in U.S. Pat. Nos. 6,620,135; 6,096,002; 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Suchneedleless devices comprising the pharmaceutical composition are alsopart of the present invention. The pharmaceutical compositions disclosedherein may also be administered by infusion. Examples of well-knownimplants and modules for administering the pharmaceutical compositionsinclude those disclosed in: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,447,233, which discloses a medicationinfusion pump for delivering medication at a precise infusion rate; U.S.Pat. No. 4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments. Many other such implants, delivery systems, and modulesare well known to those skilled in the art and those comprising thepharmaceutical compositions of the present invention are within thescope of the present invention.

Alternately, one may administer the anti-SIRPα antibody orantigen-binding fragment of the invention in a local rather thansystemic manner, for example, via injection of the antibody or fragmentdirectly into a tumor. Furthermore, one may administer the antibody orfragment in a targeted drug delivery system, for example, in a liposomecoated with a tissue-specific antibody, targeting, for example, a tumor.The liposomes will be targeted to and taken up selectively by theafflicted tissue. Such methods and liposomes are part of the presentinvention.

The administration regimen depends on several factors, including theserum or tissue turnover rate of the therapeutic antibody orantigen-binding fragment, the level of symptoms, the immunogenicity ofthe therapeutic antibody, and the accessibility of the target cells inthe biological matrix. Preferably, the administration regimen deliverssufficient therapeutic antibody or fragment to effect improvement in thetarget disease state, while simultaneously minimizing undesired sideeffects. Accordingly, the amount of biologic delivered depends in parton the particular therapeutic antibody and the severity of the conditionbeing treated. Guidance in selecting appropriate doses of therapeuticantibodies or fragments is available (see, e.g., Wawrzynczak (1996)Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina(ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, MarcelDekker, New York, N.Y.; Bach (ed.) (1993) Monoclonal Antibodies andPeptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.;Baert, et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al.(1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl.J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med.342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky etal. (2000) New Engl. J. Med. 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment. Generally, the dose begins with an amount somewhat less thanthe optimum dose and it is increased by small increments thereafteruntil the desired or optimum effect is achieved relative to any negativeside effects. Important diagnostic measures include those of symptomsof, e.g., the inflammation or level of inflammatory cytokines produced.In general, it is desirable that a biologic that will be used is derivedfrom the same species as the animal targeted for treatment, therebyminimizing any immune response to the reagent. In the case of humansubjects, for example, humanized and fully human antibodies may bedesirable.

Antibodies or antigen-binding fragments thereof disclosed herein may beprovided by continuous infusion, or by doses administered, e.g., daily,1-7 times per week, weekly, bi-weekly, monthly, bimonthly, quarterly,semiannually, annually etc. Doses may be provided, e.g., intravenously,subcutaneously, topically, orally, nasally, rectally, intramuscular,intracerebrally, intraspinally, or by inhalation. A total weekly dose isgenerally at least 0.05 μg/kg body weight, more generally at least 0.2μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.25 mg/kg, 1.0 mg/kg,2.0 mg/kg, 5.0 mg/mL, 10 mg/kg, 25 mg/kg, 50 mg/kg or more (see, e.g.,Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002)New Engl. J. Med. 346:1692-1698; Liu, et al. (1999) J. Neurol.Neurosurg. Psych. 67: 451-456; Portielji, et al. (20003) Cancer Immunol.Immunother. 52: 151-144). Doses may also be provided to achieve apre-determined target concentration of anti-SIRPα antibody in thesubject's serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg/mL or more.In other embodiments, An anti-SIRPα antibody of the present invention isadministered, e.g., subcutaneously or intravenously, on a weekly,biweekly, “every 4 weeks,” monthly, bimonthly, or quarterly basis at 10,20, 50, 80, 100, 200, 500, 1000 or 2500 mg/subject.

As used herein, the term “effective amount” refer to an amount of ananti-SIRPα or antigen-binding fragment thereof of the invention that,when administered alone or in combination with an additional therapeuticagent to a cell, tissue, or subject, is effective to cause a measurableimprovement in one or more symptoms of disease, for example cancer orthe progression of cancer. An effective dose further refers to thatamount of the antibody or fragment sufficient to result in at leastpartial amelioration of symptoms, e.g., tumor shrinkage or elimination,lack of tumor growth, increased survival time. When applied to anindividual active ingredient administered alone, an effective doserefers to that ingredient alone. When applied to a combination, aneffective dose refers to combined amounts of the active ingredients thatresult in the therapeutic effect, whether administered in combination,serially or simultaneously. An effective amount of a therapeutic willresult in an improvement of a diagnostic measure or parameter by atleast 10%; usually by at least 20%; preferably at least about 30%; morepreferably at least 40%, and most preferably by at least 50%. Aneffective amount can also result in an improvement in a subjectivemeasure in cases where subjective measures are used to assess diseaseseverity.

Kits

Further provided are kits comprising one or more components thatinclude, but are not limited to, an anti-SIRPα antibody orantigen-binding fragment, as discussed herein in association with one ormore additional components including, but not limited to apharmaceutically acceptable carrier and/or a therapeutic agent, asdiscussed herein. The antibody or fragment and/or the therapeutic agentcan be formulated as a pure composition or in combination with apharmaceutically acceptable carrier, in a pharmaceutical composition.

In one embodiment, the kit includes an anti-SIRPα antibody orantigen-binding fragment thereof of the invention or a pharmaceuticalcomposition thereof in one container (e.g., in a sterile glass orplastic vial) and/or a therapeutic agent and a pharmaceuticalcomposition thereof in another container (e.g., in a sterile glass orplastic vial).

In another embodiment, the kit comprises a combination of the invention,including an anti-SIRPα antibody or antigen-binding fragment thereof ofthe invention along with a pharmaceutically acceptable carrier,optionally in combination with one or more therapeutic agents formulatedtogether, optionally, in a pharmaceutical composition, in a single,common container.

If the kit includes a pharmaceutical composition for parenteraladministration to a subject, the kit can include a device for performingsuch administration. For example, the kit can include one or morehypodermic needles or other injection devices as discussed above.

The kit can include a package insert including information concerningthe pharmaceutical compositions and dosage forms in the kit. Generally,such information aids patients and physicians in using the enclosedpharmaceutical compositions and dosage forms effectively and safely. Forexample, the following information regarding a combination of theinvention may be supplied in the insert: pharmacokinetics,pharmacodynamics, clinical studies, efficacy parameters, indications andusage, contraindications, warnings, precautions, adverse reactions,overdosage, proper dosage and administration, how supplied, properstorage conditions, references, manufacturer/distributor information andpatent information.

The kit can also comprise a second therapeutic, for example one or moreof: anti-CD47 antibody, anti-APRIL antibody, anti-PD-1 antibody (e.g.,nivolumab, pembrolizumab, anti-PDL1 antibody, anti-TIGIT antibody,anti-CTLA4 antibody, anti-CS1 antibody (e.g., elotuzumab),anti-KIR2DL1/2/3 antibody (e.g., lirilumab), anti-CD137 antibody (e.g.,urelumab), anti-GITR antibody (e.g., TRX518), anti-PD-L1 antibody (e.g.,BMS-936559, MSB0010718C or MPDL3280A), anti-PD-L2 antibody, anti-ILT1antibody, anti-ILT2 antibody, anti-ILT3 antibody, anti-ILT4 antibody,anti-ILT5 antibody, anti-ILT6 antibody, anti-ILT7 antibody, anti-ILT8antibody, anti-CD40 antibody, anti-OX40 antibody, anti-ICOS,anti-KIR2DL1 antibody, anti-KIR2DL2/3 antibody, anti-KIR2DL4 antibody,anti-KIR2DL5A antibody, anti-KIR2DL5B antibody, anti-KIR3DL1 antibody,anti-KIR3DL2 antibody, anti-KIR3DL3 antibody, anti-NKG2A antibody,anti-NKG2C antibody, anti-NKG2E antibody, anti-4-1BB antibody (e.g.,PF-05082566), anti-TSLP antibody, anti-IL-10 antibody, IL-10 orPEGylated IL-10, or any small organic molecule inhibitor of suchtargets; an antibody or antigen binding fragment thereof binds to anantigen selected from the group consisting of AMHR2, AXL, BCMA, CA IX,CD4, CD16, CD19, CD20, CD22, CD30, CD37, CD38, CD40, CD52, CD98, CSF1R,GD2, CCR4, CS1, EpCam, EGFR, EGFRvIII, Endoglin, EPHA2, EphA3, FGFR2b,folate receptor alpha, fucosyl-GM1, HER2, HERS, IL1RAP, kappa myelomaantigen, MS4A1, prolactin receptor, TA-MUC1, and PSMA; Rituximab,ublituximab, margetuximab, IMGN-529, SCT400, veltuzumab, Obinutuzumab,ADCT-502, Hu14.18K322A, Hu3F8, Dinituximab, Trastuzumab, Cetuximab,Rituximab-RLI, c.60C3-RLI, Hu14.18-IL2, KM2812, AFM13, and (CD20)₂xCD16,erlotinib (Tarceva), daratumumab, alemtuzumab, pertuzumab, brentuximab,elotuzumab, ibritumomab, ifabotuzumab, farletuzumab, otlertuzumab,carotuximab, epratuzumab, inebilizumab, lumretuzumab, 4G7SDIE, AFM21,AFM22, LY-3022855, SNDX-6352, AFM-13, BI-836826, BMS-986012, BVX-20,mogamulizumab, ChiLob-7/4, leukotuximab, isatuximab, DS-8895, FPA144,GM102, GSK-2857916, IGN523, IT1208, ADC-1013, CAN-04, XOMA-213,PankoMab-GEX, chKM-4927, IGN003, IGN004, IGN005, MDX-1097, MOR202,MOR-208, oportuzumab, ensituximab, vedotin (Adcetris), ibritumomabtiuxetan, ABBV-838, HuMax-AXL-ADC, and ado-trastuzumab emtansine(Kadcyla); radiotherapy or chemotherapeutic agents including, but notlimited to Anthracyclines (Doxorubicin, Epirubicin, Daunorubicin,Idarubicin, Mitoxantrone), Oxaliplatin, Bortezomib, Cyclophosphamide,Bleomycin, Vorinostat, Paclitaxel, 5-Fluorouracil, Cytarabine,Prednisolone, Docetaxel, Mitomycin C, Topotecan/Camptothecin, Etoposide,Zoledronic acid, Methotrexate, Ibrutinib, Aflibercept, Bevacizumab,Toremifene, Vinblastine, Vincristine, Idelalisib, Mercaptopurine,Thalidomide, Sorafenib; a cyclic dinculeotide or other STING pathwayagonist; etc.

Detection Kits and Therapeutic Kits

As a matter of convenience, an anti-SIRPα antibody or antigen-bindingfragment thereof of the invention can be provided in a kit, i.e., apackaged combination of reagents in predetermined amounts withinstructions for performing the diagnostic or detection assay. Where theantibody or fragment is labeled with an enzyme, the kit will includesubstrates and cofactors required by the enzyme (e.g., a substrateprecursor which provides the detectable chromophore or fluorophore). Inaddition, other additives may be included such as stabilizers, buffers(e.g., a block buffer or lysis buffer) and the like. The relativeamounts of the various reagents may be varied widely to provide forconcentrations in solution of the reagents which substantially optimizethe sensitivity of the assay. Particularly, the reagents may be providedas dry powders, usually lyophilized, including excipients which ondissolution will provide a reagent solution having the appropriateconcentration.

Also provided are diagnostic or detection reagents and kits comprisingone or more such reagents for use in a variety of detection assays,including for example, immunoassays such as ELISA (sandwich-type orcompetitive format). The kit's components may be pre-attached to a solidsupport, or may be applied to the surface of a solid support when thekit is used. In some embodiments of the invention, the signal generatingmeans may come pre-associated with an antibody or fragment of theinvention or may require combination with one or more components, e.g.,buffers, antibody-enzyme conjugates, enzyme substrates, or the like,prior to use. Kits may also include additional reagents, e.g., blockingreagents for reducing nonspecific binding to the solid phase surface,washing reagents, enzyme substrates, and the like. The solid phasesurface may be in the form of a tube, a bead, a microtiter plate, amicrosphere, or other materials suitable for immobilizing proteins,peptides, or polypeptides. In particular aspects, an enzyme thatcatalyzes the formation of a chemilluminescent or chromogenic product orthe reduction of a chemilluminescent or chromogenic substrate is acomponent of the signal generating means. Such enzymes are well known inthe art. Kits may comprise any of the capture agents and detectionreagents described herein. Optionally the kit may also compriseinstructions for carrying out the methods of the invention.

Also provided is a kit comprising an anti-SIRPα antibody (e.g.,humanized antibody) or antigen-binding fragment thereof packaged in acontainer, such as a vial or bottle, and further comprising a labelattached to or packaged with the container, the label describing thecontents of the container and providing indications and/or instructionsregarding use of the contents of the container to treat one or moredisease states as described herein.

In one aspect, the kit is for treating cancer and comprises ananti-SIRPα antibody (e.g., humanized antibody) or antigen-bindingfragment thereof and a further therapeutic agent or a vaccine. The kitmay optionally further include a syringe for parenteral, e.g.,intravenous, administration. In another aspect, the kit comprises ananti-SIRPα antibody (e.g., humanized antibody) or antigen-bindingfragment thereof and a label attached to or packaged with the containerdescribing use of the antibody or fragment with the vaccine or furthertherapeutic agent. In yet another aspect, the kit comprises the vaccineor further therapeutic agent and a label attached to or packaged withthe container describing use of the vaccine or further therapeutic agentwith the anti-SIRPα antibody or fragment. In certain embodiments, ananti-SIRPα antibody and vaccine or further therapeutic agent are inseparate vials or are combined together in the same pharmaceuticalcomposition.

As discussed above in the combination therapy section, concurrentadministration of two therapeutic agents does not require that theagents be administered at the same time or by the same route, as long asthere is an overlap in the time period during which the agents areexerting their therapeutic effect. Simultaneous or sequentialadministration is contemplated, as is administration on different daysor weeks.

The therapeutic and detection kits disclosed herein may also be preparedthat comprise at least one of the antibody, peptide, antigen-bindingfragment, or polynucleotide disclosed herein and instructions for usingthe composition as a detection reagent or therapeutic agent. Containersfor use in such kits may typically comprise at least one vial, testtube, flask, bottle, syringe or other suitable container, into which oneor more of the detection and/or therapeutic composition(s) may beplaced, and preferably suitably aliquoted. Where a second therapeuticagent is also provided, the kit may also contain a second distinctcontainer into which this second detection and/or therapeuticcomposition may be placed. Alternatively, a plurality of compounds maybe prepared in a single pharmaceutical composition, and may be packagedin a single container means, such as a vial, flask, syringe, bottle, orother suitable single container. The kits disclosed herein will alsotypically include a means for containing the vial(s) in closeconfinement for commercial sale, such as, e.g., injection or blow-moldedplastic containers into which the desired vial(s) are retained. Where aradiolabel, chromogenic, fluorigenic, or other type of detectable labelor detecting means is included within the kit, the labeling agent may beprovided either in the same container as the detection or therapeuticcomposition itself, or may alternatively be placed in a second distinctcontainer means into which this second composition may be placed andsuitably aliquoted. Alternatively, the detection reagent and the labelmay be prepared in a single container means, and in most cases, the kitwill also typically include a means for containing the vial(s) in closeconfinement for commercial sale and/or convenient packaging anddelivery.

A device or apparatus for carrying out the detection or monitoringmethods described herein is also provided. Such an apparatus may includea chamber or tube into which sample can be input, a fluid handlingsystem optionally including valves or pumps to direct flow of the samplethrough the device, optionally filters to separate plasma or serum fromblood, mixing chambers for the addition of capture agents or detectionreagents, and optionally a detection device for detecting the amount ofdetectable label bound to the capture agent immunocomplex. The flow ofsample may be passive (e.g., by capillary, hydrostatic, or other forcesthat do not require further manipulation of the device once sample isapplied) or active (e.g., by application of force generated viamechanical pumps, electroosmotic pumps, centrifugal force, or increasedair pressure), or by a combination of active and passive forces.

In further embodiments, also provided is a processor, a computerreadable memory, and a routine stored on the computer readable memoryand adapted to be executed on the processor to perform any of themethods described herein. Examples of suitable computing systems,environments, and/or configurations include personal computers, servercomputers, hand-held or laptop devices, multiprocessor systems,microprocessor-based systems, set top boxes, programmable consumerelectronics, network PCs, minicomputers, mainframe computers,distributed computing environments that include any of the above systemsor devices, or any other systems known in the art.

Preferred Embodiments

Embodiment 1. An antibody or antigen binding fragment thereof that bindsto human SIRPα, wherein the antibody or antigen binding fragmentcomprises one or more, and optionally each, of:

-   -   a. a heavy chain variable region CDR1 comprising the amino acid        sequence of SEQ ID NO:69 or an amino acid sequence differing        from SEQ ID NO: 1 by 1, 2, or 3 conservative substitutions,    -   b. a heavy chain variable region CDR2 comprising the amino acid        sequence of SEQ ID NO:70 or an amino acid sequence differing        from SEQ ID NO: 2 by 1, 2, or 3 conservative substitutions,    -   c. a heavy chain variable region CDR3 comprising the amino acid        sequence of SEQ ID NO:71 or an amino acid sequence differing        from SEQ ID NO: 3 by 1, 2, or 3 conservative substitutions,    -   d. a light chain variable region CDR1 comprising the amino acid        sequence of SEQ ID NO:72 or an amino acid sequence differing        from SEQ ID NO: 4 by 1, 2, or 3 conservative substitutions,    -   e. a light chain variable region CDR2 comprising the amino acid        sequence of SEQ ID NO:73 or an amino acid sequence differing        from SEQ ID NO: 5 by 1, 2, or 3 conservative substitutions, and    -   f. a light chain variable region CDR3 comprising the amino acid        sequence of SEQ ID NO:74 or an amino acid sequence differing        from SEQ ID NO: 6 by 1, 2, or 3 conservative substitutions.        or wherein the antibody or antigen binding fragment comprises        one or more, and optionally each, of:    -   g. a heavy chain variable region CDR1 comprising the amino acid        sequence of SEQ ID NO:1 or an amino acid sequence differing from        SEQ ID NO: 1 by 1, 2, or 3 conservative substitutions,    -   h. a heavy chain variable region CDR2 comprising the amino acid        sequence of SEQ ID NO:2 or an amino acid sequence differing from        SEQ ID NO: 2 by 1, 2, or 3 conservative substitutions,    -   i. a heavy chain variable region CDR3 comprising the amino acid        sequence of SEQ ID NO:3 or an amino acid sequence differing from        SEQ ID NO: 3 by 1, 2, or 3 conservative substitutions,    -   j. a light chain variable region CDR1 comprising the amino acid        sequence of SEQ ID NO:4 or an amino acid sequence differing from        SEQ ID NO: 4 by 1, 2, or 3 conservative substitutions,    -   k. a light chain variable region CDR2 comprising the amino acid        sequence of SEQ ID NO:5 or an amino acid sequence differing from        SEQ ID NO: 5 by 1, 2, or 3 conservative substitutions, and    -   l. a light chain variable region CDR3 comprising the amino acid        sequence of SEQ ID NO:6 or an amino acid sequence differing from        SEQ ID NO: 6 by 1, 2, or 3 conservative substitutions.

Embodiment 2. The antibody or antigen binding fragment of embodiment 1,wherein the antibody or antigen binding fragment comprises

-   -   each of a heavy chain sequence comprising the amino acid        sequence of SEQ ID NO:69 or an amino acid sequence differing        from SEQ ID NO: 69 by 1, 2, or 3 conservative substitutions; the        amino acid sequence of SEQ ID NO:70 or an amino acid sequence        differing from SEQ ID NO: 70 by 1, 2, or 3 conservative        substitutions; and the amino acid sequence of SEQ ID NO: 71 or        an amino acid sequence differing from SEQ ID NO: 71 by 1, 2, or        3 conservative substitutions;        and/or    -   each of a light chain sequence comprising the amino acid        sequence of SEQ ID NO: 72 or an amino acid sequence differing        from SEQ ID NO: 72 by 1, 2, or 3 conservative substitutions; the        amino acid sequence of SEQ ID NO: 73 or an amino acid sequence        differing from SEQ ID NO: 73 by 1, 2, or 3 conservative        substitutions; and the amino acid sequence of SEQ ID NO: 74 or        an amino acid sequence differing from SEQ ID NO: 74 by 1, 2, or        3 conservative substitutions;        or wherein the antibody or antigen binding fragment comprises    -   each of a heavy chain sequence comprising the amino acid        sequence of SEQ ID NO:1 or an amino acid sequence differing from        SEQ ID NO: 1 by 1, 2, or 3 conservative substitutions; the amino        acid sequence of SEQ ID NO:2 or an amino acid sequence differing        from SEQ ID NO: 2 by 1, 2, or 3 conservative substitutions; and        the amino acid sequence of SEQ ID NO:3 or an amino acid sequence        differing from SEQ ID NO: 3 by 1, 2, or 3 conservative        substitutions;        and/or    -   each of a light chain sequence comprising the amino acid        sequence of SEQ ID NO:4 or an amino acid sequence differing from        SEQ ID NO: 4 by 1, 2, or 3 conservative substitutions; the amino        acid sequence of SEQ ID NO:5 or an amino acid sequence differing        from SEQ ID NO: 5 by 1, 2, or 3 conservative substitutions; and        the amino acid sequence of SEQ ID NO:6 or an amino acid sequence        differing from SEQ ID NO: 6 by 1, 2, or 3 conservative        substitutions.

Embodiment 3. The antibody or antigen binding fragment of embodiment 2,wherein the antibody or antigen binding fragment comprises one or bothof:

-   -   a heavy chain variable region comprising an amino acid sequence        selected from the group consisting of:        -   SEQ ID NO: 75 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 78 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 80 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 82 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 84 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 86 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 88 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto, and        -   SEQ ID NO: 102 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto;            and    -   a light chain variable region comprising an amino acid sequence        selected from the group consisting of:        -   SEQ ID NO: 76 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 90 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 92 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 94 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 96 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 98 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 100 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto, and        -   SEQ ID NO: 104 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto;            or wherein the antibody or antigen binding fragment            comprises one or both of:    -   a heavy chain variable region comprising an amino acid sequence        selected from the group consisting of:        -   SEQ ID NO: 7 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 10 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 12 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 14 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 16 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 18 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto, and        -   SEQ ID NO: 30 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto;            and    -   a light chain variable region comprising an amino acid sequence        selected from the group consisting of:        -   SEQ ID NO: 8 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 20 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 22 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 24 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto,        -   SEQ ID NO: 26 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto, and        -   SEQ ID NO: 28 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto, and        -   SEQ ID NO: 32 or an amino acid sequence at least 90%, 95%,            97%, 98%, or 99% identical thereto.

Embodiment 4. The antibody or antigen binding fragment of embodiment 3,wherein the antibody or fragment thereof has the followingcharacteristics:

-   -   binds to a cell expressing human SIRPαV1 protein with an EC₅₀<10        nM, preferably <5 nM, more preferably <1.5 nM, still more        preferably <1.0 nM, even more preferably <0.5 nM, and most        preferably about 0.3nM or less;

binds to a cell expressing human SIRPαV2 protein with an EC₅₀<10 nM,preferably <5 nM, more preferably <1.5 nM, still more preferably <1.0nM, even more preferably <0.5 nM, and most preferably about 0.3 nM orless;

-   -   does not appreciably bind to SIRPβ1 protein at an antibody        concentration of 50 nM, preferably 67 nM, and more preferably        100 nM; or alternatively at a concentration that is 10-fold        greater, preferably 50-fold greater, more preferably 100-fold        greater, and still more preferably 200-fold greater than the        antibody's EC₅₀ for SIRPαV1 or SIRPαV2;    -   inhibits binding between human SIRPα and CD47 with an IC₅₀<10.0        nM, more preferably <5.0 nM, still more preferably <2.5 nM, and        most preferably about 1.0 nM or less; and    -   exhibits a T20 “humanness” score of at least 79, and more        preferably 85.

Embodiment 5. The antibody or antigen binding fragment of embodiment 1,wherein the antibody or antigen binding fragment thereof comprises oneof the following combinations of heavy chain sequence/light chainsequence:

-   SEQ ID NO: 78/SEQ ID NO: 90,-   SEQ ID NO: 78/SEQ ID NO: 92,-   SEQ ID NO: 78/SEQ ID NO: 94,-   SEQ ID NO: 78/SEQ ID NO: 96,-   SEQ ID NO: 78/SEQ ID NO: 98,-   SEQ ID NO: 78/SEQ ID NO: 100,-   SEQ ID NO: 80/SEQ ID NO: 90,-   SEQ ID NO: 80/SEQ ID NO: 92,-   SEQ ID NO: 80/SEQ ID NO: 94,-   SEQ ID NO: 80/SEQ ID NO: 96,-   SEQ ID NO: 80/SEQ ID NO: 98,-   SEQ ID NO: 80/SEQ ID NO: 100,-   SEQ ID NO: 82/SEQ ID NO: 90,-   SEQ ID NO: 82/SEQ ID NO: 92,-   SEQ ID NO: 82/SEQ ID NO: 94,-   SEQ ID NO: 82/SEQ ID NO: 96,-   SEQ ID NO: 82/SEQ ID NO: 98,-   SEQ ID NO: 82/SEQ ID NO: 100,-   SEQ ID NO: 84/SEQ ID NO: 90,-   SEQ ID NO: 84/SEQ ID NO: 92,-   SEQ ID NO: 84/SEQ ID NO: 94,-   SEQ ID NO: 84/SEQ ID NO: 96,-   SEQ ID NO: 84/SEQ ID NO: 98,-   SEQ ID NO: 84/SEQ ID NO: 100,-   SEQ ID NO: 86/SEQ ID NO: 90,-   SEQ ID NO: 86/SEQ ID NO: 92,-   SEQ ID NO: 86/SEQ ID NO: 94,-   SEQ ID NO: 86/SEQ ID NO: 96,-   SEQ ID NO: 86/SEQ ID NO: 98,-   SEQ ID NO: 86/SEQ ID NO: 100,-   SEQ ID NO: 88/SEQ ID NO: 90,-   SEQ ID NO: 88/SEQ ID NO: 92,-   SEQ ID NO: 88/SEQ ID NO: 94,-   SEQ ID NO: 88/SEQ ID NO: 96,-   SEQ ID NO: 88/SEQ ID NO: 98,-   SEQ ID NO: 88/SEQ ID NO: 100,-   SEQ ID NO: 10/SEQ ID NO: 20,-   SEQ ID NO: 10/SEQ ID NO: 22,-   SEQ ID NO: 10/SEQ ID NO: 24,-   SEQ ID NO: 10/SEQ ID NO: 26,-   SEQ ID NO: 10/SEQ ID NO: 28,-   SEQ ID NO: 12/SEQ ID NO: 20,-   SEQ ID NO: 12/SEQ ID NO: 22,-   SEQ ID NO: 12/SEQ ID NO: 24,-   SEQ ID NO: 12/SEQ ID NO: 26,-   SEQ ID NO: 12/SEQ ID NO: 28,-   SEQ ID NO: 14/SEQ ID NO: 20,-   SEQ ID NO: 14/SEQ ID NO: 22,-   SEQ ID NO: 14/SEQ ID NO: 24,-   SEQ ID NO: 14/SEQ ID NO: 26,-   SEQ ID NO: 14/SEQ ID NO: 28,-   SEQ ID NO: 16/SEQ ID NO: 20,-   SEQ ID NO: 16/SEQ ID NO: 22,-   SEQ ID NO: 16/SEQ ID NO: 24,-   SEQ ID NO: 16/SEQ ID NO: 26,-   SEQ ID NO: 16/SEQ ID NO: 28,-   SEQ ID NO: 18/SEQ ID NO: 20,-   SEQ ID NO: 18/SEQ ID NO: 22,-   SEQ ID NO: 18/SEQ ID NO: 24,-   SEQ ID NO: 18/SEQ ID NO: 26,-   SEQ ID NO: 18/SEQ ID NO: 28,    or, in each case, at least 90%, 95%, 97%, 98%, or 99% identical to a    respective SEQ ID.

Embodiment 6. The antibody or antigen binding fragment of one ofembodiments 1-5, wherein the antibody is an intact IgG.

Embodiment 7. The antibody or antigen binding fragment of one ofembodiments 1-6, wherein the antibody comprises a wild-type or mutatedIgG2 Fc region.

Embodiment 8. The antibody or antigen binding fragment of one ofembodiments 1-6, wherein the antibody comprises a mutated IgG1 Fcregion.

Embodiment 9. The antibody or antigen binding fragment of one ofembodiments 1-6, wherein the antibody comprises a mutated IgG4 Fcregion.

Embodiment 10. An antibody or antigen binding fragment thereof thatbinds to the same epitope of human SIRPα as an antibody as an antibodyaccording to embodiment 5.

Embodiment 11. The antibody or antigen binding fragment of any ofembodiments 1-10, wherein the antibody or antigen binding fragment ishumanized.

Embodiment 12. The antibody or antigen binding fragment of any ofembodiments 1-11 that is a humanized antibody that comprises two heavychains and two light chains, wherein each heavy chain comprises SEQ IDNO: 10 and each light chain comprises SEQ ID NO: 20.

Embodiment 13. The antibody or antigen binding fragment of any ofembodiments 1-11 that is a humanized antibody that comprises two heavychains and two light chains, wherein each heavy chain comprises SEQ IDNO: 16 and each light chain comprises SEQ ID NO: 28.

Embodiment 14. The antibody or antigen binding fragment of any ofembodiments 1-11 that is a humanized antibody that comprises two heavychains and two light chains, wherein each heavy chain comprises SEQ IDNO: 18 and each light chain comprises SEQ ID NO: 20.

Embodiment 15. The antibody or antigen binding fragment of any ofembodiments 1-11 that is a humanized antibody that comprises two heavychains and two light chains, wherein each heavy chain comprises SEQ IDNO: 80 and each light chain comprises SEQ ID NO: 90.

Embodiment 16. The antibody or antigen binding fragment of any ofembodiments 1-11 that is a humanized antibody that comprises two heavychains and two light chains, wherein each heavy chain comprises SEQ IDNO: 80 and each light chain comprises SEQ ID NO: 92.

Embodiment 17. The antibody or antigen binding fragment of any ofembodiments 1-11 that is a humanized antibody that comprises two heavychains and two light chains, wherein each heavy chain comprises SEQ IDNO: 80 and each light chain comprises SEQ ID NO: 95.

Embodiment 18. The antibody or antigen binding fragment of any one ofembodiments 1-17 that comprises a glycosylation pattern characteristicof expression by a mammalian cell, and optionally is glycosylated byexpression from a CHO cell.

Embodiment 19. An isolated polypeptide comprising the amino acidsequence of any one of SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 76, 90,92, 94, 96, 98, 100, 102, 104, 7, 10, 12, 14, 16, 18, 30, 8, 20, 22, 24,26, 28, and 32, or an amino acid sequence at least 90%, 95%, 97%, 98%,or 99% identical thereto.

Embodiment 20. An isolated nucleic acid encoding any one of theantibodies or antigen binding fragments of embodiments 1-18, or any oneof the polypeptides of embodiment 19.

Embodiment 21. An isolated nucleic acid of embodiment 20 comprising:

-   -   a nucleic acid sequence of SEQ ID NO: 77 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 79 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 81 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 83 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 85 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 87 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 101 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 89 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 91 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 93 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 95 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 97 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 99 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 103 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 9 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 15 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 29 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 19 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 21 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 23 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 25 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,    -   a nucleic acid sequence of SEQ ID NO: 27 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,        and/or    -   a nucleic acid sequence of SEQ ID NO: 31 or a nucleic acid        sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

Embodiment 22. An expression vector comprising the isolated nucleic acidof embodiment 20 or 21.

Embodiment 23. An expression vector of embodiment 22, encoding both aheavy chain sequence and a light chain sequence of an anti-SIRPαantibody, the expression vectors comprising the following a firstnucleic acid sequence/second nucleic acid sequence selected from thegroup consisting of:

-   SEQ ID NO: 77/SEQ ID NO: 89,-   SEQ ID NO: 77/SEQ ID NO: 91,-   SEQ ID NO: 77/SEQ ID NO: 93,-   SEQ ID NO: 77/SEQ ID NO: 95,-   SEQ ID NO: 77/SEQ ID NO: 97,-   SEQ ID NO: 77/SEQ ID NO: 99,-   SEQ ID NO: 79/SEQ ID NO: 89,-   SEQ ID NO: 79/SEQ ID NO: 91,-   SEQ ID NO: 79/SEQ ID NO: 93,-   SEQ ID NO: 79/SEQ ID NO: 95,-   SEQ ID NO: 79/SEQ ID NO: 97,-   SEQ ID NO: 79/SEQ ID NO: 99,-   SEQ ID NO: 81/SEQ ID NO: 89,-   SEQ ID NO: 81/SEQ ID NO: 91,-   SEQ ID NO: 81/SEQ ID NO: 93,-   SEQ ID NO: 81/SEQ ID NO: 95,-   SEQ ID NO: 81/SEQ ID NO: 97,-   SEQ ID NO: 81/SEQ ID NO: 99,-   SEQ ID NO: 83/SEQ ID NO: 89,-   SEQ ID NO: 83/SEQ ID NO: 91,-   SEQ ID NO: 83/SEQ ID NO: 93,-   SEQ ID NO: 83/SEQ ID NO: 95,-   SEQ ID NO: 83/SEQ ID NO: 97,-   SEQ ID NO: 83/SEQ ID NO: 99,-   SEQ ID NO: 85/SEQ ID NO: 89,-   SEQ ID NO: 85/SEQ ID NO: 91,-   SEQ ID NO: 85/SEQ ID NO: 93,-   SEQ ID NO: 85/SEQ ID NO: 95,-   SEQ ID NO: 85/SEQ ID NO: 97,-   SEQ ID NO: 85/SEQ ID NO: 99,-   SEQ ID NO: 87/SEQ ID NO: 89,-   SEQ ID NO: 87/SEQ ID NO: 91,-   SEQ ID NO: 87/SEQ ID NO: 93,-   SEQ ID NO: 87/SEQ ID NO: 95,-   SEQ ID NO: 87/SEQ ID NO: 97,-   SEQ ID NO: 87/SEQ ID NO: 99,-   SEQ ID NO: 9/SEQ ID NO: 19,-   SEQ ID NO: 9/SEQ ID NO: 21,-   SEQ ID NO: 9/SEQ ID NO: 23,-   SEQ ID NO: 9/SEQ ID NO: 25,-   SEQ ID NO: 9/SEQ ID NO: 27,-   SEQ ID NO: 11/SEQ ID NO: 19,-   SEQ ID NO: 11/SEQ ID NO: 21,-   SEQ ID NO: 11/SEQ ID NO: 23,-   SEQ ID NO: 11/SEQ ID NO: 25,-   SEQ ID NO: 11/SEQ ID NO: 27,-   SEQ ID NO: 13/SEQ ID NO: 19,-   SEQ ID NO: 13/SEQ ID NO: 21,-   SEQ ID NO: 13/SEQ ID NO: 23,-   SEQ ID NO: 13/SEQ ID NO: 25,-   SEQ ID NO: 13/SEQ ID NO: 27,-   SEQ ID NO: 15/SEQ ID NO: 19,-   SEQ ID NO: 15/SEQ ID NO: 21,-   SEQ ID NO: 15/SEQ ID NO: 23,-   SEQ ID NO: 15/SEQ ID NO: 25,-   SEQ ID NO: 15/SEQ ID NO: 27,-   SEQ ID NO: 17/SEQ ID NO: 19,-   SEQ ID NO: 17/SEQ ID NO: 21,-   SEQ ID NO: 17/SEQ ID NO: 23,-   SEQ ID NO: 17/SEQ ID NO: 25, and-   SEQ ID NO: 17/SEQ ID NO: 27,    or, in each case, at least 90%, 95%, 97%, 98%, or 99% identical to a    respective SEQ ID NO.

Embodiment 24. A host cell comprising expression vector of embodiment 22or 23.

Embodiment 25. A host cell of embodiment 24 which produces a full lengthanti-SIRPα antibody.

Embodiment 26. The host cell of one of embodiments 24 or 25, which is abacterial cell, a human cell, a mammalian cell, a Pichia cell, a plantcell, an HEK293 cell, or a Chinese hamster ovary cell.

Embodiment 27. A composition comprising the antibody or antigen bindingfragment of any one of embodiments 1-18 and a pharmaceuticallyacceptable carrier or diluent.

Embodiment 28. The composition of embodiment 27, further comprising asecond antibody or antigen binding fragment thereof that induces ADCCand/or ADCP, wherein said antibody or antigen binding fragment of theinvention enhances the antibody-mediated destruction of cells by thesecond antibody.

Embodiment 29. The composition according to embodiment 28, wherein thesecond antibody or antigen binding fragment thereof binds to an antigenselected from the group consisting of AMHR2, AXL, BCMA, CA IX, CD4,CD16, CD19, CD20, CD22, CD30, CD37, CD38, CD40, CD52, CD98, CSF1R, GD2,CCR4, CS1, EpCam, EGFR, EGFRvIII, Endoglin, EPHA2, EphA3, FGFR2b, folatereceptor alpha, fucosyl-GM1, HER2, HERS, IL1RAP, kappa myeloma antigen,MS4A1, prolactin receptor, TA-MUC1, and PSMA.

Embodiment 30. The composition according to embodiment 29, wherein thesecond antibody or antigen binding fragment thereof is selected from thegroup consisting of Rituximab, ublituximab, margetuximab, IMGN-529,SCT400, veltuzumab, Obinutuzumab, ADCT-502, Hu14.18K322A, Hu3F8,Dinituximab, Trastuzumab, Cetuximab, Rituximab-RLI, c.60C3-RLI,Hu14.18-IL2, KM2812, AFM13, (CD20)2xCD16, erlotinib (Tarceva),daratumumab, alemtuzumab, pertuzumab, brentuximab, elotuzumab,ibritumomab, ifabotuzumab, farletuzumab, otlertuzumab, carotuximab,epratuzumab, inebilizumab, lumretuzumab, 4G7SDIE, AFM21, AFM22,LY-3022855, SNDX-6352, AFM-13, BI-836826, BMS-986012, BVX-20,mogamulizumab, ChiLob-7/4, leukotuximab, isatuximab, DS-8895, FPA144,GM102, GSK-2857916, IGN523, IT1208, ADC-1013, CAN-04, XOMA-213,PankoMab-GEX, chKM-4927, IGN003, IGN004, IGN005, MDX-1097, MOR202,MOR-208, oportuzumab, ensituximab, vedotin (Adcetris), ibritumomabtiuxetan, ABBV-838, HuMax-AXL-ADC, and ado-trastuzumab emtansine(Kadcyla).

Embodiment 31. The composition according to embodiment 28, wherein thesecond antibody or antigen binding fragment thereof induces ADCP.

Embodiment 32. The composition according to embodiment 31, wherein thesecond antibody or antigen binding fragment thereof is selected from thegroup consisting of Rituximab, ublituximab, margetuximab, IMGN-529,SCT400, veltuzumab, Obinutuzumab, Trastuzumab, Cetuximab, alemtuzumab,ibritumomab, farletuzumab, inebilizumab, lumretuzumab, 4G7SDIE,BMS-986012, BVX-20, mogamulizumab, ChiLob-7/4, GM102, GSK-2857916,PankoMab-GEX, chKM-4927, MDX-1097, MOR202, and MOR-208.

Embodiment 33. The composition of embodiment 27, further comprising oneor more agents selected from the group consisting of anti-CD27 antibody,anti-CD47 antibody, anti-APRIL antibody, anti-PD-1 antibody, anti-PD-L1antibody, anti-TIGIT antibody, anti-CTLA4 antibody, anti-CS1 antibody,anti-KIR2DL1/2/3 antibody, anti-CD137 antibody, anti-GITR antibody,anti-PD-L2 antibody, anti-ILT1 antibody, anti-ILT2 antibody, anti-ILT3antibody, anti-ILT4 antibody, anti-ILT5 antibody, anti-ILT6 antibody,anti-ILT7 antibody, anti-ILT8 antibody, anti-CD40 antibody, anti-OX40antibody, anti-ICOS, anti-KIR2DL1 antibody, anti-KIR2DL2/3 antibody,anti-KIR2DL4 antibody, anti-KIR2DL5A antibody, anti-KIR2DL5B antibody,anti-KIR3DL1 antibody, anti-KIR3DL2 antibody, anti-KIR3DL3 antibody,anti-NKG2A antibody, anti-NKG2C antibody, anti-NKG2E antibody,anti-4-1BB antibody, anti-TSLP antibody, anti-IL-10 antibody, IL-10PEGylated IL-10, an agonist (e.g., an agonistic antibody orantigen-binding fragment thereof, or a soluble fusion) of a TNF receptorprotein, an Immunoglobulin-like protein, a cytokine receptor, anintegrin, a signaling lymphocytic activation molecules (SLAM proteins),an activating NK cell receptor, a Toll like receptor, OX40, CD2, CD7,CD27, CD28, CD30, CD40, ICAM-1, LFA-1 (CD1 1a/CD18), 4-1BB (CD137),B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7,NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2Rbeta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D,ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, ITGAM, CD1 lb,ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2,TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69,SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), SLAM7, BLAME(SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, PAG/Cbp, CD19a, a ligand thatspecifically binds with CD83, inhibitor of CD47, an inhibitor of PD-1,an an inhibitor of PD-L1, an inhibitor of PD-L2, an inhibitor of CTLA4,an inhibitor of TIM3, an inhibitor of LAGS, an inhibitor of CEACAM(e.g., CEACAM-1, -3 and/or -5), an inhibitor of VISTA, an inhibitor ofBTLA, an inhibitor of TIGIT, an inhibitor of LAIR1, an inhibitor of IDO,an inhibitor of TDO, an inhibitor of CD160 an inhibitor of TGFR beta,and a cyclic dinculeotide or other STING pathway agonist.

Embodiment 34. A method of producing an antibody or antigen bindingfragment comprising:

-   -   culturing a host cell comprising a polynucleotide encoding the        heavy chain and/or the light chain of any one of the antibodies        or antigen binding fragments of embodiments 1-18 under        conditions favorable to expression of the polynucleotide; and        optionally, recovering the antibody or antigen binding fragment        from the host cell and/or culture medium.

Embodiment 35. A method for detecting the presence of a SIRPα peptide ora fragment thereof in a sample comprising contacting the sample with anantibody or fragment of any of embodiments 1-18 and detecting thepresence of a complex between the antibody or fragment and the peptide;wherein detection of the complex indicates the presence of the SIRPαpeptide.

Embodiment 36. An antibody or antigen binding fragment according to anyone of embodiments 1-18 or a composition according to any one ofembodiments 21-25, for the treatment of cancer or an infectious disease.

Embodiment 37. An antibody or antigen binding fragment of embodiments1-18 or a composition according to any one of embodiments 27-33 fordecreasing SIRPα/CD47 signalling in a human subject.

Embodiment 38. A method of treating cancer in a human subject,comprising administering to the subject an effective amount of anantibody or antigen binding fragment of any one of embodiments 1-18, oran expression vector according to one of embodiments 22 or 23, or a hostcell according to one of embodiments 24-26, or a composition accordingone of embodiments 27-33, optionally in association with a furthertherapeutic agent or therapeutic procedure.

Embodiment 39. A method of treating cancer in a human subject,comprising: administering to the subject an effective amount of

-   (i) an antibody or antigen binding fragment thereof that induces    ADCC and/or ADCP; and-   (ii) an antibody or antigen binding fragment of any one of    embodiments 1-18, or an expression vector according to one of    embodiments 22 or 23, or a host cell according to one of embodiments    24-26, or a composition according one of embodiments 27-33,    optionally in association with a further therapeutic agent or    therapeutic procedure,-   wherein the administration of (ii) enhances the antibody-mediated    destruction of cells by the antibody or antigen binding fragment    thereof that induces ADCC and/or ADCP.

Embodiment 40. The method according to embodiment 39, wherein theantibody or antigen binding fragment thereof that induces ADCC and/orADCP binds to an antigen selected from the group consisting of AMHR2,AXL, BCMA, CA IX, CD4, CD16, CD19, CD20, CD22, CD30, CD37, CD38, CD40,CD52, CD98, CSF1R, GD2, CCR4, CS1, EpCam, EGFR, EGFRvIII, Endoglin,EPHA2, EphA3, FGFR2b, folate receptor alpha, fucosyl-GM1, HER2, HERS,IL1RAP, kappa myeloma antigen, MS4A1, prolactin receptor, TA-MUC1, andPSMA.

Embodiment 41. The method according to embodiment 40, wherein theantibody or antigen binding fragment thereof that induces ADCC and/orADCP is selected from the group consisting of Rituximab, ublituximab,margetuximab, IMGN-529, SCT400, veltuzumab, Obinutuzumab, ADCT-502,Hu14.18K322A, Hu3F8, Dinituximab, Trastuzumab, Cetuximab, Rituximab-RLI,c.60C3-RLI, Hu14.18-IL2, KM2812, AFM13, (CD20)2xCD16, erlotinib(Tarceva), daratumumab, alemtuzumab, pertuzumab, brentuximab,elotuzumab, ibritumomab, ifabotuzumab, farletuzumab, otlertuzumab,carotuximab, epratuzumab, inebilizumab, lumretuzumab, 4G7SDIE, AFM21,AFM22, LY-3022855, SNDX-6352, AFM-13, BI-836826, BMS-986012, BVX-20,mogamulizumab, ChiLob-7/4, leukotuximab, isatuximab, DS-8895, FPA144,GM102, GSK-2857916, IGN523, IT1208, ADC-1013, CAN-04, XOMA-213,PankoMab-GEX, chKM-4927, IGN003, IGN004, IGN005, MDX-1097, MOR202,MOR-208, oportuzumab, ensituximab, vedotin (Adcetris), ibritumomabtiuxetan, ABBV-838, HuMax-AXL-ADC, and ado-trastuzumab emtansine(Kadcyla).

Embodiment 42. The method according to embodiment 39 or 40, wherein thesecond antibody or antigen binding fragment thereof induces ADCP.

Embodiment 43. The method according to embodiment 42, wherein the secondantibody or antigen binding fragment thereof is selected from the groupconsisting of Rituximab, ublituximab, margetuximab, IMGN-529, SCT400,veltuzumab, Obinutuzumab, Trastuzumab, Cetuximab, alemtuzumab,ibritumomab, farletuzumab, inebilizumab, lumretuzumab, 4G7SDIE,BMS-986012, BVX-20, mogamulizumab, ChiLob-7/4, GM102, GSK-2857916,PankoMab-GEX, chKM-4927, MDX-1097, MOR202, and MOR-208.

Embodiment 44. A method of treating an infection or infectious diseasein a human subject, comprising administering to the subject an effectiveamount of an antibody or antigen binding fragment of any one ofembodiments 1-18, or an expression vector according to one ofembodiments 22 or 23, or a host cell according to one of embodiments24-26, or a composition according one of embodiments 27-33, optionallyin association with a further therapeutic agent or therapeuticprocedure.

Embodiment 45. An antibody having one or more of the followingcharacteristics:

-   -   binds human SIRPαV1 protein having the sequence of SEQ ID NO: 34        with an EC_(50 <1) nM; exhibits at least a 100-fold higher EC₅₀        for SIRPαV1(P74A) having the sequence of SEQ ID NO: 62; and        exhibits at least a 100-fold higher EC₅₀ for human SIRPβ1        protein having the sequence of SEQ ID NO: 38, preferably when        measured by cellular ELISA;    -   binds to a cell expressing human SIRPαV1 protein with an EC₅₀<10        nM, preferably <5 nM, more preferably <1.5 nM, still more        preferably <1.0 nM, even more preferably <0.5 nM, and most        preferably about 0.3 nM or less;    -   binds to a cell expressing human SIRPαV2 protein with an EC₅₀<10        nM, preferably <5 nM, more preferably <1.5 nM, still more        preferably <1.0 nM, even more preferably <0.5 nM, and most        preferably about 0.3nM or less;

does not appreciably bind to SIRPβ1 protein at an antibody concentrationof 50 nM, preferably 67 nM, and more preferably 100 nM; or alternativelyat a concentration that is 10-fold greater, preferably 50-fold greater,more preferably 100-fold greater, and still more preferably 200-foldgreater than the antibody's EC₅₀ for SIRPαV1 or SIRPαV2;

-   -   inhibits binding between human SIRPα and CD47 with an IC₅₀<10.0        nM, more preferably <5.0 nM, still more preferably <2.5 nM, and        most preferably about 1.0 nM or less; and    -   exhibits a T20 “humanness” score of at least 79, and more        preferably 85.

Embodiment 46. The antibody or antigen binding fragment of embodiment 45that binds human SIRPαV1 protein having the sequence of SEQ ID NO: 34with an EC₅₀<1 nM; exhibits at least a 100-fold higher EC₅₀ forSIRPαV1(P74A) having the sequence of SEQ ID NO: 62; and exhibits atleast a 100-fold higher EC₅₀ for human SIRPβ1 protein having thesequence of SEQ ID NO: 38.

Embodiment 47. The antibody or antigen binding fragment of embodiment 45or 46 that comprises one or two light chains comprising SEQ ID NO: 20 ora sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto and oneor two heavy chains comprising SEQ ID NO: 10 or a sequence at least 90%,95%, 97%, 98%, or 99% identical thereto.

Embodiment 48. The antibody or antigen binding fragment of embodiment 45or 46 that comprises one or two light chains comprising SEQ ID NO: 28 ora sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto and oneor two heavy chains comprising SEQ ID NO: 16 or a sequence at least 90%,95%, 97%, 98%, or 99% identical thereto.

Embodiment 49. The antibody or antigen binding fragment of embodiment 45or 46 that comprises one or two light chains comprising SEQ ID NO: 20 ora sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto and andone or two heavy chains comprising SEQ ID NO: 18 or a sequence at least90%, 95%, 97%, 98%, or 99% identical thereto.

Embodiment 50. The antibody or antigen binding fragment of embodiment 45or 46 that comprises one or two light chains comprising SEQ ID NO: 90 ora sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto and oneor two heavy chains comprising SEQ ID NO: 80 or a sequence at least 90%,95%, 97%, 98%, or 99% identical thereto.

Embodiment 51. The antibody or antigen binding fragment of embodiment 45or 46 that comprises one or two light chains comprising SEQ ID NO: 92 ora sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto and oneor two heavy chains comprising SEQ ID NO: 80 or a sequence at least 90%,95%, 97%, 98%, or 99% identical thereto.

Embodiment 52. The antibody or antigen binding fragment of embodiment 45or 46 that comprises one or two light chains comprising SEQ ID NO: 96 ora sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto and andone or two heavy chains comprising SEQ ID NO: 80 or a sequence at least90%, 95%, 97%, 98%, or 99% identical thereto.

Embodiment 53. The antibody or antigen binding fragment of one ofembodiments 45-52, wherein the antibody is an intact IgG.

Embodiment 54. The antibody or antigen binding fragment of one ofembodiments 45-52, wherein the antibody comprises a wild-type or mutatedIgG2 Fc region.

Embodiment 55. The antibody or antigen binding fragment of one ofembodiments 45-52, wherein the antibody comprises a mutated IgG1 Fcregion.

Embodiment 56. The antibody or antigen binding fragment of one ofembodiments 45-52, wherein the antibody comprises a mutated IgG4 Fcregion.

Embodiment 57. An antibody or antigen binding fragment thereof thatbinds to the same epitope of human SIRPα as an antibody as an antibodyaccording to one of embodiments 45-52.

Embodiment 58. The antibody or antigen binding fragment of any ofembodiments 45-52, wherein the antibody or antigen binding fragment ishumanized.

Embodiment 59. A composition comprising the antibody or antigen bindingfragment of any one of embodiments 45-52 and a pharmaceuticallyacceptable carrier or diluent.

Embodiment 60. An antibody or antigen binding fragment according to anyone of embodiments 45-52 or a composition according to embodiment 59,for the treatment of cancer or an infectious disease.

Embodiment 61. An antibody or antigen binding fragment according to anyone of embodiments 45-52 or a composition according to embodiment 59 fordecreasing SIRPα/CD47 signalling in a human subject.

Embodiment 62. A method of treating cancer in a human subject,comprising administering to the subject an effective amount of anantibody or antigen binding fragment according to any one of embodiments45-52 or a composition according to embodiment 59, optionally inassociation with a further therapeutic agent or therapeutic procedure.

Embodiment 63. A method of treating an infection or infectious diseasein a human subject, comprising administering to the subject an effectiveamount of an antibody or antigen binding fragment according to any oneof embodiments 45-52 or a composition according to embodiment 59,optionally in association with a further therapeutic agent ortherapeutic procedure.

General Methods

Standard methods in molecular biology are described Sambrook, Fritschand Maniatis (1982 & 1989 2^(nd) Edition, 2001 3^(rd) Edition) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Sambrook and Russell (2001) Molecular Cloning,3^(rd) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego,Calif.). Standard methods also appear in Ausbel, et al. (2001) CurrentProtocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. NewYork, N.Y., which describes cloning in bacterial cells and DNAmutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2),glycoconjugates and protein expression (Vol. 3), and bioinformatics(Vol. 4).

Methods for protein purification including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization aredescribed (Coligan, et al. (2000) Current Protocols in Protein Science,Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis,chemical modification, post-translational modification, production offusion proteins, glycosylation of proteins are described (see, e.g.,Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2,John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) CurrentProtocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY,N.Y., pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for LifeScience Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech(2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production,purification, and fragmentation of polyclonal and monoclonal antibodiesare described (Coligan, et al. (2001) Current Protcols in Immunology,Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999)Using Antibodies, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Harlow and Lane, supra). Standard techniques forcharacterizing ligand/receptor interactions are available (see, e.g.,Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, JohnWiley, Inc., New York).

Monoclonal, polyclonal, and humanized antibodies can be prepared (see,e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ.Press, New York, N.Y.; Kontermann and Dubel (eds.) (2001) AntibodyEngineering, Springer-Verlag, New York; Harlow and Lane (1988)Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., pp. 139-243; Carpenter, et al. (2000) J.Immunol. 165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang et al.(1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem.272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote andWinter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

An alternative to humanization is to use human antibody librariesdisplayed on phage or human antibody libraries in transgenic mice(Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995)Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377;Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Kay et al. (1996)Phage Display of Peptides and Proteins: A Laboratory Manual, AcademicPress, San Diego, Calif.; de Bruin et al. (1999) Nature Biotechnol.17:397-399).

Single chain antibodies and diabodies are described (see, e.g., Maleckiet al. (2002) Proc. Natl. Acad. Sci. USA 99:213-218; Conrath et al.(2001) J. Biol. Chem. 276:7346-7350; Desmyter et al. (2001) J. Biol.Chem. 276:26285-26290; Hudson and Kortt (1999) J. Immunol. Methods231:177-189; and U.S. Pat. No. 4,946,778). Bifunctional antibodies areprovided (see, e.g., Mack, et al. (1995) Proc. Natl. Acad. Sci. USA92:7021-7025; Carter (2001) J. Immunol. Methods 248:7-15; Volkel, et al.(2001) Protein Engineering 14:815-823; Segal, et al. (2001) J. Immunol.Methods 248:1-6; Brennan, et al. (1985) Science 229:81-83; Raso, et al.(1997) J. Biol. Chem. 272:27623; Morrison (1985) Science 229:1202-1207;Traunecker, et al. (1991) EMBO J. 10:3655-3659; and U.S. Pat. Nos.5,932,448, 5,532,210, and 6,129,914).

Bispecific antibodies are also provided (see, e.g., Azzoni et al. (1998)J. Immunol. 161:3493; Kita et al. (1999) J. Immunol. 162:6901; Merchantet al. (2000) J. Biol. Chem. 74:9115; Pandey et al. (2000) J. Biol.Chem. 275:38633; Zheng et al. (2001) J. Biol Chem. 276:12999; Propst etal. (2000) J. Immunol. 165:2214; Long (1999) Ann. Rev. Immunol. 17:875).Purification of antigen is not necessary for the generation ofantibodies. Animals can be immunized with cells bearing the antigen ofinterest. Splenocytes can then be isolated from the immunized animals,and the splenocytes can fused with a myeloma cell line to produce ahybridoma (see, e.g., Meyaard et al. (1997) Immunity 7:283-290; Wrightet al. (2000) Immunity 13:233-242; Preston et al., supra; Kaithamana etal. (1999) J. Immunol. 163:5157-5164).

Antibodies can be conjugated, e.g., to small drug molecules, enzymes,liposomes, polyethylene glycol (PEG). Antibodies are useful fortherapeutic, diagnostic, kit or other purposes, and include antibodiescoupled, e.g., to dyes, radioisotopes, enzymes, or metals, e.g.,colloidal gold (see, e.g., Le Doussal et al. (1991) J. Immunol.146:169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsingand Bishop (1999) J. Immunol. 162:2804-2811; Everts et al. (2002) J.Immunol. 168:883-889).

Methods for flow cytometry, including fluorescence activated cellsorting (FACS), are available (see, e.g., Owens, et al. (1994) FlowCytometry Principles for Clinical Laboratory Practice, John Wiley andSons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2^(nd) ed.;Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry, JohnWiley and Sons, Hoboken, N.J.). Fluorescent reagents suitable formodifying nucleic acids, including nucleic acid primers and probes,polypeptides, and antibodies, for use, e.g., as diagnostic reagents, areavailable (Molecular Probes (2003) Catalogue, Molecular Probes, Inc.,Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

Standard methods of histology of the immune system are described (see,e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology andPathology, Springer Verlag, New York, N.Y.; Hiatt, et al. (2000) ColorAtlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.;Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, NewYork, N.Y.).

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments, are available (see, e.g.,GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, Md.); GCG WisconsinPackage (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp.,Crystal Bay, Nev.); Menne, et al. (2000) Bioinformatics 16: 741-742;Menne, et al. (2000) Bioinformatics Applications Note 16:741-742; Wren,et al. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res.14:4683-4690).

EXAMPLES

The following examples serve to illustrate the present invention. Theseexamples are in no way intended to limit the scope of the invention.

Example 1: Specificity of Commercial hSIRPα Antibodies

The specificity of various commercially available monoclonal anti-hSIRPαantibodies (Table 7) for binding to hSIRPα variant 1 (hSIRPαV1; GenBankaccession: NM_001040022.1) (SEQ ID NO: 34), hSIRPα variant 2 (hSIRPαV2;GenBank accession: D86043.1) (SEQ ID NO: 36), hSIRPβ1 (GenBankaccession: NM_006065.4) (SEQ ID NO: 38), hSIRPβ1 transcript variant3/hSIRPβL (NCBI accession: NM_001135844.3) (SEQ ID NO: 117), and hSIRPγ(NCBI accession: NM_018556.3) (SEQ ID NO: 40) was evaluated by cellularELISA (CELISA). Reactivity was confirmed using CHO-K1 cells (ATCCCCL-61) that had been transiently transfected, using Lipofectamine 2000,with cDNA encoding the full length open reading frame of hSIRPαV1,hSIRPαV2, hSIRPβ1, hSIRPβL, and hSIRPγ subcloned into the pCI-neo vector(Promega, Madison, Wis.). CHO-K1.hSIRPαV1, CHO-K1.hSIRPαV2,CHO-K1.hSIRPβ1, CHO-K1.hSIRPβL, and CHO-K1.hSIRPγ cells were seeded inculture medium (DMEM-F12 (Gibco) supplemented with 5% New Born CalfSerum (BioWest) and Pen/Strep (Gibco)) in 96-well flat-bottom tissueculture plates and incubated at 37° C., 5% CO₂ and 95% humidity for 24hours. Subsequently, culture medium was removed and cells were incubatedfor 1 hour at 37° C., 5% CO₂ and 95% humidity with purified hSIRPγantibodies (used at 10 μg/mL and dilutions thereof). Next, cells werewashed with PBS-T and incubated for 1 hour at 37° C., 5% CO₂ and 95%humidity with goat-anti-mouse IgG-HRP (Southern Biotech). Subsequently,cells were washed three times with PBS-T and immunoreactivity againsthSIRPαV1, hSIRPαV2, hSIRPβ1, hSIRPβL, and hSIRPγ was visualized with TMBStabilized Chromogen (Invitrogen). Reactions were stopped with 0.5 MH₂SO₄ and absorbances were read at 450 and 610 nm. EC50 values, theconcentration at which 50% of the total binding signal is observed, werecalculated using GraphPad Prism 6 (GraphPad Software, Inc.).

TABLE 7 Commercially available hSIRPα antibodies used for comparisonwith antibodies generated herein. Target Clone Cat.# Company SpeciesReactivity Isotype hSIRPα SE5A5 323802 Biolegend mouse human IgG1 hSIRPα7B3 LS-C340387 LifeSpan Biosciences mouse human IgG1 hSIRPα 1B5LS-C338479 LifeSpan Biosciences mouse human IgG1 hSIRPα 1C6 LS-C338477LifeSpan Biosciences mouse human IgG1 hSIRPα 27 sc-136067 Santa Cruzmouse human, mouse, rat IgG1 Biotechnology hSIRPα SE7C2 sc-23863 SantaCruz mouse human IgG1 Biotechnology hSIRPα P3C4 LS-C179629-100CliniSciences mouse human IgG2a hSIRPα 2A4A5 W172-3 MBL Internationalmouse human IgG2a hSIRPα 15-414 LS-C58098 LifeSpan Biosciences mousehuman IgG2a hSIRPα 1H1 LS-C338476 LifeSpan Biosciences mouse human IgG2ahSIRPα C-7 sc-376884 Santa Cruz mouse human IgG2a Biotechnology hSIRPα03 11612-MM03-100 Sino Biological Inc. mouse human IgG2b hSIRPα 5E10 LSC83566 LifeSpan Biosciences mouse human IgG2b hSIRPα 602411 MAB4546 R&Dmouse human IgG2b hSIRPα EPR16264 ab191419 Abcam rabbit human, mouse,rat IgG hSIRPα D6I3M 13379S Cell Signaling rabbit human, mouse, rat, IgGTechnology monkey hSIRPα 001 50956- Sino Biological Inc. rabbit mouse,human IgG R001_100 ug hSIRPα REA144 130-099-768 Miltenyi Biotec humanhuman IgG1 hSIRPα KWAR23 TAB-453CT Creative Biolabs human human IgG4

As depicted in FIG. 1 and the following Table 8, commercially availablehSIRPα antibodies cross-react with at least hSIRPβ1, hSIRPβL, or hSIRPγor demonstrate allele-specific binding to hSIRPαV2. The KWAR23 antibodycross-reacts with all members of the SIRP receptor family tested: itbinds to hSIRPαV1, hSIRPαV2, hSIRPβ1, hSIRPβL, and hSIRPγ.

TABLE 8 hSIRPαV1 hSIRPαV2 hSIRPI31 hSIRPy hSIRPI3L binding bindingbinding binding binding EC50 EC50 EC50 EC50 EC50 Antibody (nM) (nM) (nM)(nM) (nM) hSIRPα.50A 1.626 1.627 nd 1.475 0.639 anti-hSIRPα (cloneSE5A5) 0.372 0.186 0.185 0.200 0.122 anti-hSIRPα (clone 7B3) 0.187 0.3000.255 nd 0.206 anti-hSIRPα (clone 1B5) nd 0.122 nd nd nd anti-hSIRPα(clone 106) 0.739 0.167 2.965 15.589 2.008 anti-hSIRPα (clone 27) nd ndnd nd nd anti-hSIRPα (clone SE7C2) 1.269* 0.300 nd 1.525 26.818*anti-hSIRPα (clone P3C4) 0.288 2.154 0.383 0.365 0.136 anti-hSIRPα(clone 2A4A5) nd 1.005 8.633 nd 12.156* anti-hSIRPα (clone 15-414) nd ndnd nd nd anti-hSIRPα (clone 1H1) nd 0.204 nd nd nd anti-hSIRPα (cloneC-7) nd nd nd nd nd anti-hSIRPα (clone 03) 96.016* 15.059* 16.043*17.303* 9.109* anti-hSIRPα (clone 5E10) nd nd nd nd nd anti-hSIRPα(clone 602411) 0.068 nd 0.081 3.622 0.060 anti-hSIRPα (clone EPR16264)nd 2.450* nd nd nd anti-hSIRPα (clone D6I3M) 18.690* 8.762* nd nd ndanti-hSIRPα (clone 001) 18.081* nd nd 0.494 6.253* anti-hSIRPα (cloneREA144) 5.243* 3.274* 4.534* 3.212* 2.147* KWAR23 0.067 0.062 0.1400.043 0.097 Values indicated with * were extrapolated; nd, not detected

Example 2: Immunization and Selection of Anti-hSIRPα Antibodies

To generate SIRPα antibodies that bind to all known SIRPα alleles andare not binding SIRPβ1 mice were immunized with a pCI-neo expressionconstruct encoding hSIRPαV1 and hSIRPαV2. Mice were immunized by genegun immunization using a Helios Gene gun (BioRad, Hercules, Calif.) andDNA coated gold bullets (BioRad) following manufacturer's instructions.Briefly, 1 μm gold particles were coated with pCI-neo-hSIRPαV1 orpCI-neo-hSIRPαV2 cDNA and commercial expression vectors for mouse Flt3Land mouse GM-CSF in a 2:1:1 ratio (both from Aldevron, Fargo, ND). Atotal of 1 μg of plasmid DNA was used to coat 500 μg of gold particles.Specifically, 7-8 weeks old female BALB/C mice (Harlan) were immunizedin the ears with a gene gun, receiving 3 administration cycles in bothears.

For positive and negative B-cell selection and CELISA purposes,CHO-K1.hSIRPαV1, CHO-K1.hSIRPαV2, CHO-K1.hSIRPβ1, and CHO-K1.hCD47stable cell lines were generated by transfecting CHO-K1 cells withpCI-neo vector encoding the full length open reading frame of hSIRPαV1,hSIRPαV2, hSIRPβ1, and hCD47 (NCBI accession: NM_001777.3) (SEQ ID NO:42), respectively. Stable clones were obtained by limiting dilution.

Antibody titer was assessed by CELISA, using the CHO-K1.hSIRPαV1 andCHO-K1.hSIRPαV2 stable cell lines. These hSIRPα-expressing CHO-K1 celllines were maintained in DMEM-F12 (Gibco) supplemented with 10% FetalBovine Serum (Hyclone) and 80U Pen/Strep (Gibco). Cells were seeded into96-well flat-bottom tissue culture plates at 8×10⁴ cells/well andcultured at 37° C., 5% CO₂ and 95% humidity until cell layers wereconfluent. Cells were incubated with each sample of the diluted mousesera for 1 hour at 37° C., 5% CO₂ and 95% humidity. Next, cells werewashed with Phosphate buffered Saline (PBS)/0.05% Tween-20 (PBS-T) andincubated with goat-anti-mouse IgG-HRP conjugate (Southern Biotech) for1 hour at 37° C., 5% CO₂ and 95% humidity. Subsequently, cells werewashed three times with PBS-T and anti-hSIRPα immunoreactivity wasvisualized with TMB Stabilized Chromogen (Invitrogen). Reactions werestopped with 0.5 M H₂SO₄ and absorbances were read at 450 and 610 nm.The anti-hSIRPα titer was higher than 1:2,500 in each individual mouseserum sample as detected after two DNA immunizations. All mice thatdemonstrated reactivity against hSIRPαV1 and hSIRPαV2 were immunized fora final, third time and sacrificed 14 days later. Erythrocyte-depletedspleen and lymph-node cell populations were prepared as describedpreviously (Steenbakkers et al., 1992, J. Immunol. Meth. 152: 69-77;Steenbakkers et al., 1994, Mol. Biol. Rep. 19: 125-134) and frozen at−180° C.

To select anti-hSIRPα antibody producing B-cells, a selection strategywas designed and developed that preferentially bound B-cells expressingantibodies that bind to hSIRPαV1 and hSIRPαV2. Splenocytes and lymphnodes were harvested from the hSIRPαV1/V2 immunized mice and isolatedcells were incubated with CHO-K1.hSIRPβ1 that were seeded into T25culture flasks and irradiated at 30 Gray. After 1 hour unbound cellswere gently removed by moving the flask back and forth. Mediumcontaining unbound cells was then transferred to a new T25 flaskcontaining irradiated CHO-K1.hSIRPβ1 cells. This procedure was followedfor in total three times on ice in order to negatively selecthSIRPβ1-reactive B-cells. Next, medium containing unbound B-cells wasincubated with CHO-K1.hSIRPαV1 and CHO-K1.hSIRPαV2 cells that wereirradiated at 3,000 Gray. After 1.5 hours incubation on ice unboundcells were removed with multiple wash steps using culture medium.Subsequently, T25 flasks containing CHO-K1.hSIRPαV1 and CHO-K1.hSIRPαV2cells with bound lymphocytes were harvested with Trypsin-EDTA (Sigma).Bound B-cells were cultured, as described by Steenbakkers et al., 1994,Mol. Biol. Rep. 19: 125-134. Briefly, selected B-cells were mixed with10% (v/v) T-cell supernatant and 50,000 irradiated (25 Gray) EL-4 B5feeder cells in a final volume of 200 μl medium in 96-well flat-bottomtissue culture plates. On day eight, supernatants were screened forhSIRPαV1 and hSIRPαV2 reactivity by CELISA as described below.

CHO-K1.hSIRPαV1, CHO-K1.hSIRPαV2, and CHO-K1.hSIRPβ1 were seeded inculture medium (DMEM-F12 (Gibco) supplemented with 10% Fetal BovineSerum (Hyclone) and 80U Pen/Strep (Gibco)) in 96-well flat-bottom tissueculture plates and cultured at 37° C., 5% CO₂ and 95% humidity untilthey were confluent. Subsequently, culture medium was removed and cellswere incubated for 1 hour at 37° C., 5% CO₂ and 95% humidity withsupernatants from the B-cell cultures. Next, cells were washed withPBS-T and incubated for 1 hour at 37° C., 5% CO₂ and 95% humidity withgoat-anti-mouse IgG-HRP conjugate (Southern Biotech). Subsequently,cells were washed three times with PBS-T and anti-hSIRPαV1,anti-hSIRPαV2, and anti-hSIRPβ1 immunoreactivity was visualized with TMBStabilized Chromogen (Invitrogen). Reactions were stopped with 0.5 MH₂SO₄ and absorbances were read at 450 and 610 nm.

Immunoreactivity to human SIRPγ was assessed by ELISA using recombinanthSIRPγ/Fc-protein (R&D Systems, Cat. #4486-SB-050; SEQ ID NO: 108)coated 96-well MaxiSorp flat-bottom plates. Protein coated 96-wellplates were blocked in PBS/1% bovine serum albumin (BSA) for 1 hour atroom temperature (RT). PBS/1% BSA was removed and plates were incubatedfor 1 hour at RT with supernatants from the B-cell cultures. Next,plates were washed with PBS-T and incubated for 1 hour at RT withgoat-anti-mouse IgG-HRP conjugate (Southern Biotech). Subsequently,wells were washed three times with PBS-T and anti-hSIRPγimmunoreactivity was visualized with TMB Stabilized Chromogen(Invitrogen). Reactions were stopped with 0.5 M H₂SO₄ and absorbanceswere read at 450 and 610 nm.

B-cell clones from the hSIRPα reactive supernatants, which were not orwhich were minimally reactive to hSIRPβ1 were immortalized bymini-electrofusion following published procedures (Steenbakkers et al.,1992, J. Immunol. Meth. 152: 69-77; Steenbakkers et al., 1994, Mol.Biol. Rep. 19:125-34) with some minor deviations (e.g. pronase reactionwas omitted). Briefly, B-cells were mixed with 10⁶ Sp2/0-Ag14 murinemyeloma cells (ATCC CRL-1581) in Electrofusion Isomolar Buffer(Eppendorf). Electrofusions were performed in a 50 μL fusion chamber byan alternating electric field of 15 s, 1 MHz, 23 Vrms AC followed by asquare, high field DC pulse of 10 μs, 180 Volt DC and again by analternating electric field of 15 s, 1 MHz, 23 Vrms AC. Content of thechamber was transferred to hybridoma selective medium and plated in a96-well plate under limiting dilution conditions. On day 10 followingthe electrofusion, hybridoma supernatants were screened for hSIRPαV1,hSIRPαV2, hSIRPβ1, and hSIRPγ binding activity by CELISA and ELISA, asdescribed above. Hybridomas that secreted antibodies in the supernatantthat specifically bound hSIRPαV1 and hSIRPαV2 were both frozen at −180°C. (−1 batch) and subcloned by limited dilution to safeguard theirintegrity and stability. Stable hybridomas were frozen at −180° C. (−LD1batch) until cell layers were confluent.

Further selection of the hybridomas was performed by assessing theblocking abilities of the hSIRPαV1/hCD47 interaction in CELISA format.For the assessment of hCD47 blockade CHO-K1.hCD47 cells were seeded in384-well flat-bottom tissue culture plates and incubated at 37° C., 5%CO₂ and 95% humidity in culture medium. Recombinant hSIRPα/Fc-protein(R&D Systems, Cat. #4546-SA-050; SEQ ID NO: 107) was pre-incubated witha dilution series of the hybridoma supernatants containing hSIRPαreactive antibodies and control antibodies (at 10 μg/mL and dilutionsthereof) for 30 minutes at 37° C., 5% CO₂ and 95% humidity. ConfluentCHO-K1.hCD47 cells were washed with PBS-T and incubated for 1 hour withthe mixtures containing hSIRPα reactive antibodies and recombinanthSIRPα/Fc-protein at 37° C., 5% CO₂ and 95% humidity. Next, cells werewashed with PBS-T followed by addition of goat-anti-human IgG-HRPconjugate (Jackson Immuno Research) to the cells, which was incubatedfor 1 hour at 37° C., 5% CO₂ and 95% humidity. Subsequently cells werewashed three times with PBS-T and binding of hSIRPα/Fc-protein wasvisualized with TMB Stabilized Chromogen (Invitrogen). Reactions werestopped with 0.5 M H₂SO₄ and absorbances were read at 450 and 610 nm.

Selected stable hybridomas were cultured in serum-free media for 7 days;supernatants were harvested and antibodies were purified using MabSelectSure Protein A resin according to the manufacturer's instructions (GEHealthcare). Antibody concentrations were quantified usingspectrophotometry. Supernatants of the hybridoma cultures were used toisotype the hybridomas. In short, isotyping was done using a mousemonoclonal antibody isotyping kit (Biorad) based on a dipstick withimmobilized goat-anti-mouse antibody bands to each of the common mouseisotypes and light chains. Recovered antibodies were all identified asmouse IgG1. Antibody sequences were elucidated by sequencing of variableregions of the mouse IgG1 hybridoma material performed at LakePharma,using the following method: the total RNA of the hybridoma cells wasextracted, which allowed cDNA synthesis. Rapid Amplification of cDNAEnds (RACE) was performed that allowed cloning of positive fragments ina TOPO (Thermo Fisher Scientific) vector. TOPO clones were sequenced andsequences were annotated using VBASE2 (Retter et al., VBASE2, anintegrative V gene database. Nucleic Acids Res. 2005 Jan. 1; 33(Databaseissue):D671-4).

Example 3: Characterization of hSIRPα Antibodies

The binding specificity of antibody hSIRPα.50A to hSIRPα was comparedantibody KWAR23 (Canadian Patent 2939293 A1), in a CELISA format. CHO-K1cells were transiently transfected with hSIRPαV1, hSIRPαV2, hSIRPβ1, andhSIRPγ (GenBank accession: NM_018556.3) (SEQ ID NO: 39) cDNAs.Subsequently, hSIRPα binding was assessed by CELISA usingCHO-K1.hSIRPαV1, CHO-K1.hSIRPαV2, CHO-Kl.hSIRPβ1, and CHO-K1.hSIRPγcells. Detection of bound antibody was performed with goat-anti-mouseIgG-HRP (Southern Biotech) for mouse antibodies including hSIRPα.50A andcontrol antibodies or, alternatively, with goat-anti-human IgG-HRPconjugate (Jackson Immuno Research) for the KWAR23 antibody. KWAR23 (SEQID NO: 130; SEQ ID NO: 131) was expressed as a chimeric human IgG4 kappaantibody in CHO cells. As shown in FIG. 2 and the following Table 9,KWAR23 antibody cross-reacts with all members of the SIRP receptorfamily tested: it binds to hSIRPαV1, hSIRPαV2, hSIRPβ1, and hSIRPy. EC₅₀values represent the concentration at which 50% of the total bindingsignal is observed (average and SD were calculated from values of twoindependent experiments).

TABLE 9 hSIRPαV1 binding ECSO (nM) hSIRPαV2 binding ECSO (nM) AntibodyAverage SD Average SD KWAR23 0,081 0,001 0,051 0,004 hSIRPα.50A 1,3650,164 1,296 0,186 anti-hSIRPα (clone SESAS) 0,304 0,200 anti-hSIRPy(clone LSB2.20) nd nd hSIRP−binding ECM) (nM) hSIRPy binding ECM) (nM)Antibody Average SD Average SD KWAR23 0,161 0,007 0,040 0,002 hSIRPα.50And nd 1,249 0,179 anti-hSIRPα (clone SE5A5) 0,192 0,168 anti-hSIRPy(clone LSB2.20) nd 0,265 Empty squares indicate n=measurements. nd, notdetected

In addition, the specificity of hSIRPα.50A for all known of hSIRPαalleles (allelic variants as described by Takenaka et al., 2007, NatImmunol. 8:1313-1323) was further investigated by CELISA using the samestrategy as above. To this end, hSIRPα.50A binding was assessed usingCHO-K1 cells that were transiently transfected with cDNAs encoding fulllength hSIRPαV1, hSIRPαV2, hSIRPαV3 (NA07056_V3) (SEQ ID NO: 43),hSIRPαV4 (NA11832_V4) (SEQ ID NO: 45), hSIRPαV5 (NA18502_V5) (SEQ ID NO:47), hSIRPαV6 (NA18507_V6) (SEQ ID NO: 49), hSIRPαV8 (NA18570_V8) (SEQID NO: 51), and hSIRPαV9 (NA18943_V9) (SEQ ID NO: 53). FIG. 3 and thefollowing Table 10 demonstrate the reactivity of antibody clonehSIRPα.50A for each of these hSIRPα alleles. EC50 values represent theconcentration at which 50% of the total binding signal is observed(average and SD were calculated from values of two independentexperiments).

TABLE 10 Antibody anti-hSIRPα hSIRPα.50A (clone SE5A5) hSIRPαV1 EC50(nM) 0,936 0,327 SD 0,285 0,107 hSIRPαV2 EC50 (nM) 0,665 0,200 SD 0,1060,046 hSIRPαV3 EC50 (nM) 0,688 0,226 SD 0,097 0,052 hSIRPαV4 EC50 (nM)0,824 0,256 SD 0,280 0,085 hSIRPαV5 EC50 (nM) 0,765 0,276 SD 0,210 0,086hSIRPαV6 EC50 (nM) 0,954 0,098 SD 0,437 0,050 hSIRPαV8 EC50 (nM) 0,6440,300 SD 0,066 0,061 hSIRPαV9 EC50 (nM) 0,733 0,260 SD 0,205 0,079

Example 4: hCD47 Blocking Ability of hSIRPα.50A

The hSIRPα.50A antibody was analyzed by flow cytometry for its abilityto block recombinant hCD47/Fc-protein (R&D Systems, Cat.# 4670-CD-050;SEQ ID NO: 109) binding to cell surface expressed hSIRPα. For thispurpose, THP-1 (ATCC TIB-202) and U-937 (ATCC CRL-1593.2) monocyte celllines were used as the source of hSIRPα in the assay. THP-1 and U-937cells were seeded in 96-well round bottomed tissue culture plates andincubated for 45 minutes with FcR Blocking Reagent (Miltenyi Biotec) andhSIRPα.50A antibody (200 μg/mL and dilutions thereof) in PBS/1% BSA at4° C. Next, cells were washed three times with PBS/1% BSA and incubatedwith DyLight 488-labeled recombinant hCD47/Fc-protein for 30 minutes at4° C. After this labeling procedure, cells were washed two times,resuspended in PBS/1% BSA containing 0.1 μg/mL DAPI (BioLegend), andanalysed by flow cytometry on the FACSCanto II (BD Biosciences). Datawere processed and analysed with FlowJo V10 software (FlowJo, LLC).

As depicted in FIG. 4 and the following Table 11, binding of recombinanthCD47 fused to an Fc domain of human IgG1 was monitored in the presenceof increasing amounts of the hSIRPα.50A antibody. Antibody hSIRPα.50Ablocked the hSIRPα/hCD47 interaction, using the flow cytometry-basedmethod described above. IC50 values for the blockade of hCD47 werecalculated from this data. IC50 values represent the concentration atwhich half of the inhibition is observed.

TABLE 11 THP-1 U-937 Antibody IC50 (nM) IC50 (nM) hSIRPα.50A 4,605 7,164

Next, the binding of hSIRPα.50A to hSIRPα expressed on primary humanCD14⁺ monocytes was investigated. In addition, the ability of hSIRPα.50Ato block the interaction between hSIRPα and recombinant hCD47/Fc-proteinwas assessed. For this purpose, CD14+ monocytes were isolated fromFicoll-purified human peripheral blood mononuclear cells (PBMCs) usingRosetteSep human monocyte enrichment cocktail (Stemcell). The percentageof monocytes present after the enrichment was determined by flowcytometry on the FACSVerse (BD Biosciences) based on CD14 staining usingan APC-Cy7-conjugated mouse-anti-human CD14 detection antibody (BDBiosciences). Subsequently, CD14+ enriched PBMCs were seeded in 96-wellround bottomed tissue culture plates and incubated for 40 minutes withFcR Blocking Reagent (Miltenyi Biotec) containing hSIRPα.50A antibody(25 μg/mL and dilutions thereof) in PBS/1% BSA at 4° C. Next, cells werewashed three times with PBS/1% BSA and incubated with a FITC-labeledgoat-anti-mouse Ig (BD Biosciences) detection antibody in PBS/1% BSA for40 minutes at 4° C. After this labeling procedure, cells were washed twotimes, resuspended in PBS/1% BSA containing 0.1 μg/mL DAPI (BioLegend),and analysed by flow cytometry on the FACSVerse (BD Biosciences). Datawere processed and analysed with FlowJo V10 software (FlowJo, LLC).

FIGS. 5A and B and the following Table 12 indicates that hSIRPα.50Abinds to primary human CD14+ enriched monocytes. EC₅₀ values representthe concentration at which 50% of the total binding signal is observed.To assess the blocking ability of hSIRPα.50A, CD14+ enriched monocytescells were seeded in 96-well round bottomed tissue culture plates andincubated for 45 minutes with FcR Blocking Reagent (Miltenyi Biotec) andhSIRPα.50A antibody (200 μg/mL and dilutions thereof) in PBS/1% BSA at4° C. Thereafter, cells were washed three times with PBS/1% BSA andincubated with 10 μg/mL DyLight 488-labeled recombinant hCD47/Fc-proteinfor 45 minutes at 4° C. After this labeling procedure, cells were washedtwo times, resuspended in PBS/1% BSA containing 0.1 μg/mL DAPI(BioLegend), and analysed by flow cytometry on the FACSVerse (BDBiosciences). Data were processed and analysed with FlowJo V10 software(FlowJo, LLC). FIGS. 5C and D and the following Table 12 demonstratesthe ability of antibody hSIRPα.50A to block the hSIRPα/hCD47interaction. IC50 values for the blockade of hCD47 were calculated fromthis data. IC50 values represent the concentration at which half of theinhibition is observed.

TABLE 12 Donor 1 Donor 2 Antibody EC50 (nM) IC50 (nM) EC50 (nM) IC50(nM) hSIRPα.50A 7,381 4,618 3,081 1,035

Example 5: Functionality of hSIRPα.50A mAb in the Human GranulocytePhagocytosis Assay

To confirm the functionality of hSIRPα.50A in primary immune cells,granulocytes (e.g. effector cells) were isolated from healthy humandonor EDTA blood. First, the EDTA blood of each donor was pooled andcentrifuged at 300 g for 6 minutes at 20° C. Next, plasma was removed byaspiration, and the remaining blood cells were gently resuspended. Cellswere recovered in red blood cell (RBC) lysis buffer (155 mM NH4Cl; 10 mMKHCO3) and incubated for 10 minutes on ice. Next, cells were centrifugedat 300 g for 7 minutes. Supernatants containing lysed RBCs were removedby aspiration, and the remaining blood cells were gently resuspended inRBC lysis buffer and kept on ice for 1 minute. RBC lysis was neutralizedby adding assay medium (IMDM (Gibco) supplemented with 10% Fetal BovineSerum (Gibco) and Pen/Strep (Gibco)). Blood cells were centrifuged at300 g for 6 minutes and supernatants were removed by aspiration toremove remaining RBCs as much as possible. Subsequently,erythrocyte-lysed blood cells were resuspended in assay mediumcontaining 10 ng/mL IFNγ and cells were incubated for 1 hour at 37° C.,5% CO₂ and 95% humidity. Non-adherent blood cells containing humangranulocytes were collected by mild washing of the tissue culture platewith assay medium (monocytes are depleted due to adherence to theplastic surface). The percentage of granulocytes present in the cellsuspension was determined by flow cytometry on the FACSCanto II (BDBiosciences) based on high forward scatter (FSC) and side scatter (SSC).Binding of hSIRPα.50A to human granulocytes was assessed by incubatingthe cells for 30 minutes at 4° C. with hSIRPα.50A antibody (25 μg/mL anddilutions thereof) in PBS/1% BSA containing 10% autologous serum (PBS/1%BSA/10% serum). Next, cells were washed three times with PBS/1% BSA/10%serum and incubated for 30 minutes at 4° C. with a FITC-labeledgoat-anti-mouse Ig (BD Biosciences) detection antibody. After thislabeling procedure, cells were washed two times, resuspended in PBS/1%BSA/10% serum and analysed by flow cytometry on the FACSCanto II (BDBiosciences). Data were processed and analysed with FlowJo V10 software(FlowJo, LLC). FIG. 6A shows that hSIRPα.50A binds to primary humangranulocytes. EC50 values represent the concentration at which 50% ofthe total binding signal is observed.

Next, target cells were fluorescently labeled with either cellproliferation dye eFluor450 (eBioscience) in the case of Raji (ECACC85011429), Daudi (ECACC 85011437), Ramos (ECACC 85030802), and BJAB(DSMZ ACC-757) lymphoma cells or, alternatively, with Vybrant DiDcell-labeling solution (Thermo Fisher Scientific) for FaDu cells.Labeling was performed according to manufacturer's instructions. Labeledtarget cells were co-cultured for 2-3 hours at 37° C., 5% CO₂ and 95%humidity with isolated primary human granulocytes in a 1:1 ratio(7.5*10⁴ cells of each target and effector per well of a 96-well roundbottomed tissue culture plate) in the presence of 0.1 μg/mL rituximab(anti-hCD20). In addition, cells were co-cultured with 0.1 μg/mLrituximab in presence of 10 μg/mL hSIRPα.50A. Phagocytosis was assayedby determining the percentage of granulocytes positive for eFluor450 (orDID) using flow cytometry on the FACSCanto II (BD Biosciences). Datawere processed and analysed with FlowJo V10 software (FlowJo, LLC).

Compared to the mouse IgG1 isotype control, hSIRPα.50A potently enhancestumor cell phagocytosis induced by rituximab (FIG. 6B). The sameprocedure was followed with other existing therapeutic antibodies suchas 0.05 μg/mL daratumumab (anti-hCD38), 0.1 μg/mL alemtuzumab(anti-hCD52), and 0.1 μg/mL cetuximab (anti-hEGFR) (FIG. 6C-E). Thesedata demonstrate that hSIRPα.50A enhances antibody-mediated tumor cellphagocytosis by human granulocytes.

Example 6: Functionality of hSIRPα.50A mAb in the Human MacrophagePhagocytosis Assay

Blockade of CD47 by hSIRPα.50A enhances the phagocytosis of humanlymphoma cells tumor cells by human macrophages. Human macrophages weregenerated by first enriching CD14+ monocytes from Ficoll-purified humanperipheral blood mononuclear cells (PBMCs) using RosetteSep humanmonocyte enrichment cocktail (Stemcell). Monocytes were seeded intoCellCarrier 96-well flat-bottom microplates (Perkin Elmer) and culturedin macrophage medium (IMDM (Gibco) supplemented with 8.5% Fetal BovineSerum (Gibco) and Pen/Strep (Gibco)) containing 50 ng/mL human monocytecolony stimulating factor (M-CSF) for 7 days at 37° C., 5% CO₂ and 95%humidity to promote differentiation into macrophages. Thesemonocyte-derived macrophages (MDMs) become adherent allowing other cellsto be washed away. Human Raji, Daudi, Ramos, and BJAB lymphoma cellswere counted and labeled with cell proliferation dye eFluor450(eBioscience) following manufacturer's instructions. After labeling, thelymphoma cells were mixed with assay medium (RPMI (Gibco) supplementedwith 10% Fetal Bovine Serum (Gibco) and Pen/Strep (Gibco)) containing 10μg/mL anti-hSIRPα antibodies, respective isotype controls and either 0.1μg/mL rituximab (anti-hCD20) or 0.05 μg/mL daratumumab (anti-hCD38). Thelymphoma cells were then added to the individual wells containing MDMsat a ratio of 2.5:1 tumor cells per phagocyte, mixed and incubated at37° C., 5% CO₂ and 95% humidity for 2 hours. After the incubation, thewells were washed with PBS to remove most of the non-phagocytosed tumorcells, and cells were fixed with 2% formaldehyde for 10 min at RT. Thewells were then washed and maintained in PBS/3% BSA in dark at 4° C.overnight. Lymphoma cells present in the wells were stained withbiotin-conjugated anti-human CD19 clone HIB19 (eBioscience) for 1 hourat RT, and subsequently were counterstained with Alexa Fluor488-conjugated streptavidin (Thermo Fisher Scientific) for 1 hour at RT.Next, nuclei were stained with DRAQS (Thermo Fisher Scientific) for 10minutes at RT, mixture was removed, and PBS was added to each well.Cells were analysed with the Operetta automated fluorescence microscope(Perkin Elmer). Data were processed and analysed with Columbus V2.6software.

As shown in FIG. 7, hSIRPα.50A enhances rituximab anddaratumumab-mediated phagocytosis activity. The phagocytosis of humanlymphoma cells was quantified using a phagocytosis index, as follows:(number of tumor cells inside macrophages/number of macrophages)*100;counting at least 200 macrophages per sample.

Example 7: Humanized Antibody Design and CDR Grafting

The mouse hSIRPα.50A antibody was humanized using CDR-graftingtechnology (see e.g. U.S. Pat. No. 5,225,539 and Williams, D. G. et al.,2010, Antibody Engineering, volume 1, Chapter 21).

First, human germline sequences were identified using IgBLAST (Ye J. etal., 2013, Nucleic Acids Res. 41:W34-40). For the hSIRPα.50A VH humangermline sequence, V-gene IGHV1/OR15-2*02 was identified (75.2%identity) and for the VL human germline sequence IGKV1-27*01 wasidentified (74.0% identity). These two germline sequences were used todirectly graft the mouse CDRs, resulting in the following two cDNAconstructs: SEQ ID NO: 17 (VH) and SEQ ID NO: 25 (VL).

Next, a database was constructed containing all human sequencesavailable in the IMGT database (Lefranc, M.-P. et al., 1999, NucleicAcid Res. 27:209-212) identifying 85,848 individual sequences. Thesesequences were queried using TBLASTN (2.2.31+) to identify templatesequences that demonstrated the highest identify to the framework ofhSIRPα.50A VH and VL sequences. Three VH and three VL sequences wereidentified that demonstrated a similarity score of 75% or higher andthat displayed similar CDR lengths, preferably identical to those inhSIRPα.50A VH CDR1, CDR2, CDR3 and VL CDR1, CDR2 and CDR3, respectively.

For the heavy chain, the frameworks encoded by GenBank (Benson, D. A. etal., 2013, Nucleic Acids Res. 41(D1): D36-42) accession # AB066948,AB067235, and U84168 were selected as templates for straight grafting ofthe hSIRPα.50A VH CDRs, resulting in the following cDNA constructs: SEQID NO: 9, 11 and 13, respectively. For the light chain, the frameworksencoded by GenBank accession #JF894288, AB363321, and L12101 wereselected as templates for straight grafting of the hSIRPα.50A VL CDRs,resulting in the following cDNA constructs: SEQ ID NO: 19, 21 and 23.Framework and CDR definition were those as described by Kabat et al.(“Sequences of Proteins of Immunological Interest”, Kabat, E., et al.,US Department of Health and Human Services, (1983)).

To understand the effect of humanized framework residues on thestructure of the Fv, a homology model of the mouse hSIRPα.50A Fv wasmade using the ‘Antibody Modeling Cascade’ (default parameters) withinDiscovery Studio 4.5. The homology model was built on basis of PDB ID1CIC, for the light chain and Fv, and PDB ID 4Q0X for the heavy chain.The CDRs were grafted in silico to study residues that are close to anyof the CDRs and which might affect the loop conformation, referred asVernier residues. Residues that might affect the loop conformation, andwhich are within <5 Å to the CDR surface were identified and substitutedwith the mouse amino acid at this position. The resulting templates werechecked for the presence of post translational modification (PTM) motifsusing Discovery Studio 4.5 and where possible (i.e. non-CDR, non-Vernierresidues) changed to prevent a PTM. For the heavy chain, removal of thepredicted sequence PTM motifs and structural considerations (i.e.rigidity of the backbone) in the hSIRPα.50A VH resulted in the design ofone additional construct: SEQ ID NO: 15. For the light chain the PTMremoval resulted in the following construct: SEQ ID NO: 27.

CDRs were grafted on each of the identified templates, expressed as ahuman IgG4 (SEQ ID NO: 65), kappa (SEQ ID NO: 63) antibody cloned in thepcDNA3.1(+) vector (Thermo Fisher Scientific) and for transienttransfection in FreeStyle 293-F human embryonic kidney cells(HEK293T/17, ATCC CRL-11268). In each case, an IgG4 version carrying thestabilizing Adair mutation (Angal S. et al., 1993, Mol Immunol. 30:105-108), where Serine 228 is converted to Proline, was used.

Example 8: Synthesis, Expression and Purification of HumanizedConstructs

Plasmids encoding the heavy chain and light chain constructs were mixedin a 1:1 ratio (30 μg in total) and transiently expressed bytransfection into FreeStyle 293-F cells using 293fectin transfectionreagent (Invitrogen) following the manufacturer's instructions.Supernatants (30 ml) were harvested after 7 days and antibodies werepurified using MabSelect Sure Protein A resin according to themanufacturer's instructions (GE Healthcare). Buffer was exchanged for 10mM Histidine, 100 mM NaCl pH 5.5 buffer using Zeba desalting columns(Thermo Fisher Scientific). The concentration of purified antibodies wasdetermined based on OD280 (Nanodrop ND-1000). Endotoxin level wasdetermined by LAL-test according to the manufacturer's instructions(Lonza).

Example 9: Binding of Humanized SIRPα Antibodies

Binding of the humanized antibodies to hSIRPγ was studied in CELISAformat. Binding of the hSIRPγ antibodies to human SIRPαV1, SIRPαV2,hSIRPβ1, and hSIRPγ was confirmed using CHO-K1 cells that had beentransiently transfected with cDNA encoding the full length open readingframe of each of these respective targets subcloned into the pCI-neovector. CHO-K1.hSIRPαV1, CHO-K1.hSIRPαV2, CHO-K1.hSIRPβ1, andCHO-K1.hSIRPγ cells were seeded in culture medium (DMEM-F12 (Gibco)supplemented with 5% New Born Calf Serum (BioWest) and Pen/Strep(Gibco)) in 96-well flat-bottom tissue culture plates and incubated at37° C., 5% CO₂ and 95% humidity until cell layers were confluent.Subsequently, culture medium was removed and cells were incubated for 1hour at 37° C., 5% CO₂ and 95% humidity with purified hSIRPγ antibodies(10 μg/mL and dilutions thereof). Next, cells were washed with PBS-T andincubated for 1 hour at 37° C., 5% CO₂ and 95% humidity withgoat-anti-human IgG-HRP conjugate (Jackson Immuno Research) orgoat-anti-mouse IgG-HRP (Southern Biotech). Subsequently, cells werewashed three times with PBS-T and anti-hSIRPγ immunoreactivity wasvisualized with TMB Stabilized Chromogen (Invitrogen). Reactions werestopped with 0.5 M H₂SO₄ and absorbances were read at 450 and 610 nm.EC50 values, the concentration at which 50% of the total binding signalis observed, were calculated using GraphPad Prism 6 (GraphPad Software,Inc.). In Table 13 the EC50 values of the humanized hSIRPγ antibodiesare depicted.

TABLE 13 Binding of humanized and parental hSIRPα.50A antibodies to CHO-K1.hSIRPαV1, CHO-K1.hSIRPαV2, CHO-K1.hSIRPβ1, and CHO-K1.hSIRPγ cells.EC50 values represent the concentration at which 50% of the totalbinding signal is observed (average and SD were calculated from valuesof two independent experiments). hSIRPαV1 binding EC50 hSIRPαV2 bindingEC50 hSIRPβ1 binding EC50 hSIRPγ binding EC50 (nM) (nM) (nM) (nM)Antibody Average SD Average SD Average SD Average SD hSIRPα.50H 0.8830.212 0.864 0.109 nd nd 1.485* 0.120 1L1 hSIRPα.50H 0.781 0.104 0.8160.161 nd nd 1.259* 0.155 1L2 hSIRPα.50H 1.094 0.112 1.107 0.238 nd nd2.579* 0.672 1L3 hSIRPα.50H 1.488 0.259 1.621 0.320 nd nd 7.435* 0.2081L4 hSIRPα.50H 0.962 0.235 0.848 0.239 nd nd 1.013* 0.115 1L5 hSIRPα.50H1.097 0.286 1.056 0.303 nd nd 1.424* 0.080 3L1 hSIRPα.50H 1.055 0.3470.999 0.450 nd nd 1.502* 0.305 3L2 hSIRPα.50H 1.159 0.417 1.160 0.429 ndnd 2.471* 0.530 3L3 hSIRPα.50H 1.261 0.317 1.520 0.333 nd nd 5.175*0.210 3L4 hSIRPα.50H 0.878 0.097 0.868 0.190 nd nd 1.199* 0.120 3L5hSIRPα.50H 0.683 0.027 0.681 0.156 nd nd 0.950* 0.171 4L1 hSIRPα.50H0.737 0.110 0.651 0.147 nd nd 0.871* 0.062 4L2 hSIRPα.50H 0.933 0.0780.898 0.133 nd nd 1.596* 0.144 4L3 hSIRPα.50H 1.197 0.175 1.240 0.238 ndnd 1.980* 0.681 4L4 hSIRPα.50H 0.701 0.136 0.661 0.161 nd nd 0.808*0.038 4L5 hSIRPα.50H 0.731 0.039 0.709 0.063 nd nd 1.028* 0.087 5L1hSIRPα.50H 0.675 0.086 0.572 0.023 nd nd 0.822* 0.046 5L2 hSIRPα.50H1.029 0.084 0.796 0.004 nd nd 1.612* 0.247 5L3 hSIRPα.50H 1.169 0.1971.115 0.060 nd nd 4.028* 0.342 5L4 hSIRPα.50H 0.681 0.066 0.611 0.030 ndnd 0.868* 0.028 5L5 hSIRPα.50A 1.365 0.164 1.296 0.186 nd nd 1.249*0.179 Note that variants with the H2 heavy chain could not be expressedin FreeStyle 293-F cells; values indicated with * were extrapolated; nd,not detected

Binding of the parental and humanized hSIRPγ antibodies to hSIRPγ wasfurther assessed using NK-92MI cells (ATCC CRL-2408), an interleukin-2(IL-2) independent natural killer cell line derived from the NK-92 cellline. NK-92MI cells were seeded in 96-well round bottomed tissue cultureplates and incubated for 30 minutes with the humanized hSIRPα.50Aantibody variants (100 μg/mL and dilutions thereof) in PBS/1% BSA at 4°C. Next, cells were washed three times with PBS/1% BSA and incubated for30 minutes at 4° C. with a FITC-labeled mouse-anti-human IgG4 (Abcam) ordonkey-anti-mouse IgG (Jackson Immuno Research) detection antibody inPBS/1% BSA. After this labeling procedure, cells were washed two times,resuspended in PBS/1% BSA and analysed by flow cytometry on theFACSCanto II (BD Biosciences). Data were processed and analysed withFlowJo V10 software (FlowJo, LLC).

Example 10: Blockade of hCD47 Binding to hSIRPγ by Humanized hSIRPα.50AAntibodies

hCD47 blockade was assessed by flow cytometry for the full panel ofhumanized hSIRPα.50A antibodies. To this end, HEK293 cells (ATCCCRL-1573) were transiently transfected using Lipofectamine 2000(Invitrogen) with the pCI-neo vector encoding the full length openreading frame of human SIRPαV1. The transfected cells were cultured at37° C., 5% CO₂ and 95% humidity in medium (DMEM-F12 (Gibco) with 10%Fetal Bovine Serum (Gibco) and Pen/Strep (Gibco)) until confluent.Subsequently, cells were dissociated and seeded in 96-well roundbottomed tissue culture plates and incubated for 30 minutes with thehumanized hSIRPα.50A antibody variants (100 μg/mL and dilutions thereof)in PBS/1% BSA at 4° C. Next, cells were washed three times with PBS/1%BSA and incubated with recombinant hCD47/Fc-protein (ModiQuest; SEQ IDNO: 42) for 30 minutes at 4° C. Afterwards, cells were washed threetimes with PBS/1% BSA and incubated for 30 minutes at 4° C. with amouse-anti-human IgG1 Hinge-FITC (Southern Biotech) detection antibody.After this labeling procedure, cells were washed two times, resuspendedin PBS/1% BSA and analysed by flow cytometry on the FACSCanto II (BDBiosciences). Data were processed and analysed with FlowJo V10 software(FlowJo, LLC) and plotted using GraphPad Prism 6 (GraphPad Software,Inc.) (FIG. 8).

As depicted in FIG. 8, binding of recombinant hCD47 fused to an Fcdomain of human IgG1 was monitored in the presence of increasing amountsof the humanized hSIRPα.50A antibody variants. All antibody variantsblocked the hSIRPα/hCD47 interaction.

Example 11: Binding Domain of hSIRPα.50A

To identify the binding region of hSIRPα.50A, several SIRPαexchange-mutants were designed based on the human SIRPαV1 and hSIRPβ1amino acid sequence. Based on the fold of SIRPα, the extracellularregion can be subdivided into three separate domains: the Ig-like(immunoglobulin-like) V-type (IgV), Ig-like C1-type (IgC1), and Ig-likeC2-type (IgC2) domain. The IgV domain is also known as theligand-binding N-terminal domain of SIRPα (which binds to CD47). Thehuman SIRPαV1/β1 mutants were designed on the basis of the full lengthhSIRPαV1 sequence (SEQ ID NO: 33) and each individual Ig-like domain wassubstituted for the equivalent domain of human SIRPβ1 (SEQ ID NO: 37).The cDNAs encoding the constructs, hSIRPα-VβC1αC2α (SEQ ID NO: 55),hSIRPα-VαC1βC2α (SEQ ID NO: 57), and hSIRPα-VαC1αC2β (SEQ ID NO: 59)were synthesized (GeneArt) and subcloned into the pCI-neo vector.Binding of hSIRPα.50A to the exchange mutants was tested using CELISA.To this end, CHO-Kl cells were transiently transfected, usingLipofectamine 2000, with the pCI-neo vectors encoding hSIRPαV1,hSIRPαV2, hSIRPβ1, hSIRPα-VβC1αC2α, hSIRPα-VαC1βC2α, andhSIRPα-VαC1αC2β, respectively. The transfected cells were cultured at37° C., 5% CO₂ and 95% humidity in medium (DMEM-F12 (Gibco) with 5% NewBorn Calf serum (Biowest) and Pen/Strep (Gibco)) until confluent.Subsequently, cells were trypsinized and seeded in 96-well flat-bottomtissue culture plates and cultured at 37° C., 5% CO₂ and 95% humidity inculture medium until confluent. Then, culture medium was removed andcells were incubated for 1 hour at 37° C., 5% CO₂ and 95% humidity withhSIRPα.50A and anti-hSIRPα clone SE5A5 antibodies. Next, cells werewashed with PBS-T and incubated for 1 hour at 37° C., 5% CO₂ and 95%humidity with goat-anti-mouse IgG-HRP conjugate (Southern Biotech).After that, cells were washed three times with PBS-T and anti-hSIRPαimmunoreactivity was visualized with TMB Stabilized Chromogen(Invitrogen). Reactions were stopped with 0.5 M H₂SO₄ and absorbanceswere read at 450 and 610 nm.

The antibody of the present invention demonstrated loss of binding tothe hSIRPα-VβC1αC2α mutant, indicating that hSIRPα.50A binds to the IgVdomain of hSIRPα (FIG. 9; Table 14). EC50 values represent theconcentration at which 50% of the total binding signal is observed(average and SD were calculated from values of two independentexperiments).

TABLE 14 Antibody anti-hSIRPα hSIRPα.50A (clone SE5A5) hSIRPαV1 EC50(nM) 0,321 0,117 SD 0,018 0,001 hSIRPαV2 EC50 (nM) 0,215 0,084 SD 0,0120,012 hSIRPβ1 EC50 (nM) nd 0,180 SD nd 0,025 hSIRPα-VβC1aC2α EC50 (nM)nd 0,121 SD nd 0,003 hSIRPα-VαC1βC2α EC50 (nM) 0,345 0,135 SD 0,0080,013 hSIRPα-VαC1αC2β 13 EC50 (nM) 0,408 0,127 SD 0,039 0,028

To pinpoint the amino acids for interaction of hSIRPα.50A with the IgVdomain, several point mutants of hSIRPαV1 were generated based on singleamino acid differences between hSIRPαV1/V2 and hSIRPβ1. FIG. 10A showsan alignment of the hSIRPα and hSIRPβ1 IgV domain. Amino acids in thehSIRPα IgV domain that are altered in hSIRPβ1 were mutated by using theQuikChange II Site-Directed Mutagenesis Kit (Stratagene) and the fulllength hSIRPαV1 sequence (SEQ ID NO: 33) as donor cDNA. Binding ofhSIRPα.50A to hSIRPαV1 point mutants was tested using CELISA. To thisend, CHO-K1 cells were transiently transfected, using Lipofectamine2000, with cDNA encoding the full length open reading frame of hSIRPαV1and mutants thereof, and hSIRPβ1 subcloned into the pCI-neo vector.Transfected cells were seeded in culture medium (DMEM-F12 (Gibco)supplemented with 5% New Born Calf Serum (BioWest) and Pen/Strep(Gibco)) in 96-well flat-bottom tissue culture plates and incubated at37° C., 5% CO₂ and 95% humidity for 24 hours. Subsequently, culturemedium was removed and cells were incubated for 1 hour at 37° C., 5% CO₂and 95% humidity with purified hSIRPα antibodies (used at 10 μg/mL anddilutions thereof). Next, cells were washed with PBS-T and incubated for1 hour at 37° C., 5% CO₂ and 95% humidity with goat-anti-mouse IgG-HRP(Southern Biotech). Subsequently, cells were washed three times withPBS-T and immunoreactivity against hSIRPαV1, hSIRPαV1 mutants, andhSIRPβ1 was visualized with TMB Stabilized Chromogen (Invitrogen).Reactions were stopped with 0.5 M H₂SO₄ and absorbances were read at 450and 610 nm. EC50 values, the concentration at which 50% of the totalbinding signal is observed, were calculated using GraphPad Prism 6(GraphPad Software, Inc.) (average and SD were calculated from values oftwo independent experiments). As shown in FIG. 10B and the followingTable 15, the Proline at position 74 (P74) constitues a crucial aminoacid for the specific binding of hSIRPα.50A to hSIRPαV1. Expression ofhSIRPαV1(P74A) (SEQ ID NO: 61), where P74 is converted to Alanine, onCHO-K1 cells results in loss of hSIRPα.50A antibody binding. Thisproline is absent in the IgV domain sequence of hSIRPβ1.

TABLE 15 hSIRPαV1 binding EC50 (nM) hSIRPβ1 binding EC50 (nM)hSIRPαV1(P74A) binding EC50 (nM) Antibody Average SD Average SD AverageSD hSIRPα.50A 0.535 0.152 nd nd nd nd anti-hSIRPα (clone SE5A5) 0.1640.008 0.156 0.009 0.150 0.013

Example 12: Characterization of hSIRPα.40A and hSIRPα.50A Antibodies

The binding specificity of antibodies hSIRPα.40A and hSIRPα.50A tohSIRPα were compared in a CELISA format. In short, CHO-K1 cells weretransiently transfected with hSIRPαV1, hSIRPαV2, hSIRPβ1, hSIRPβL, andhSIRPγ cDNAs. Subsequently, hSIRPα binding was assessed by CELISA usingCHO-K1.hSIRPαV1, CHO-K1.hSIRPαV2, CHO-K1.hSIRPβ1, CHO-K1.hSIRPβL, andCHO-K1.hSIRPγ cells. Detection of bound antibody was done withgoat-anti-mouse IgG-HRP (Southern Biotech). As shown in FIG. 11 and thefollowing Table 16, hSIRPα.40A and hSIRPα.50A antibodies bind tohSIRPαV1, hSIRPαV2, hSIRPβL, and hSIRPγ, but do not display detectablehSIRPβ1 binding. EC₅₀ values represent the concentration at which 50% ofthe total binding signal is observed (average and SD were calculatedfrom values of two independent experiments).

TABLE 16 hSIRPαV1 binding hSIRPαV2 binding hSIRPβ1 binding hSIRPγbinding hSIRPβL binding EC50 (nM) EC50 (nM) EC50 (nM) EC50 (nM) EC50(nM) Antibody Average SD Average SD Average SD Average SD Average SDhSIRPα.40A 0.109 0.036 0.088 0.002 nd nd 0.099 0.055 0.141 0.078hSIRPα.50A 1.428 0.371 1.156 0.127 nd nd 1.990 0.827 0.632 0.277 nd, notdetected

In addition, the specificity of hSIRPα.40A for all known of hSIRPαalleles (allelic variants as described by Takenaka et al., Nat Immunol.8:1313-1323 (2007) was further investigated by CELISA using the samestrategy as above. To this end, hSIRPα.40A binding was assessed usingCHO-K1 cells that were transiently transfected with cDNAs encoding fulllength hSIRPαV1, hSIRPαV2, hSIRPαV3 (NA07056_V3) (SEQ ID NO: 44),hSIRPαV4 (NA11832_V4) (SEQ ID NO: 46), hSIRPαV5 (NA18502_V5) (SEQ ID NO:48), hSIRPαV6 (NA18507_V6) (SEQ ID NO: 50), hSIRPαV8 (NA18570_V8) (SEQID NO: 52), and hSIRPαV9 (NA18943_V9) (SEQ ID NO: 54). FIG. 12 and thefollowing Table 17 demonstrates the reactivity of antibody clonehSIRPα.40A for each of these hSIRPα alleles. EC₅₀ values represent theconcentration at which 50% of the total binding signal is observed(average and SD were calculated from values of two independentexperiments).

TABLE 17 Antibody hSIRPα.40A hSIRPα.50A hSIRPαV1 EC50 (nM) 0.134 1.690hSIRPαV2 EC50 (nM) 0.089 1.066 hSIRPαV3 EC50 (nM) 0.107 1.767 hSIRPαV4EC50 (nM) 0.100 1.297 hSIRPαV5 EC50 (nM) 0.115 1.260 hSIRPαV6 EC50 (nM)0.136 2.219 hSIRPαV8 EC50 (nM) 0.089 1.508 hSIRPαV9 EC50 (nM) 0.1151.367

Example 13: hCD47 Blocking Ability of hSIRPα.40A

The hSIRPα.40A antibody was analyzed by flow cytometry for its abilityto block recombinant hCD47/Fc-protein (R&D Systems, Cat. #4670-CD-050;SEQ ID NO: 109) binding to cell surface expressed hSIRPα. For thispurpose, THP-1 (ATCC TIB-202) and U-937 (ATCC CRL-1593.2) monocyte celllines were used as the source of hSIRPα in the assay. THP-1 and U-937cells were seeded in 96-well round bottomed tissue culture plates andincubated for 45 minutes with FcR Blocking Reagent (Miltenyi Biotec) andhSIRPα.40A antibody (100 μg/mL and dilutions thereof) in PBS/1% BSA at4° C. Next, cells were washed three times with PBS/1% BSA and incubatedwith DyLight 488-labeled recombinant hCD47/Fc-protein for 30 minutes at4° C. After this labeling procedure, cells were washed two times,resuspended in PBS/1% BSA containing 0.1 μg/mL DAPI (BioLegend), andanalysed by flow cytometry on the FACSCanto II (BD Biosciences). Datawere processed and analysed with FlowJo V10 software (FlowJo, LLC).

As depicted in FIG. 13 and the following Table 18, binding ofrecombinant hCD47 fused to an Fc domain of human IgG1 was monitored inthe presence of increasing amounts of the hSIRPα.40A antibody. AntibodyhSIRPα.40A blocked the hSIRPα/hCD47 interaction, using the flowcytometry-based method described above. IC50 values for the blockade ofhCD47 were calculated from this data. IC50 values represent theconcentration at which half of the inhibition is observed.

TABLE 18 THP-1 IC50 U-937 Antibody (nM) IC50 (nM) hSIRPα.40A 0.646 1.344hSIRPα.50A 7.833 19.501

Next, the binding of hSIRPα.40A to hSIRPα expressed on primary humanCD14⁺ monocytes was investigated. In addition, the ability of hSIRPα.40Ato block the interaction between hSIRPα and recombinant hCD47/Fc-proteinwas assessed. For this purpose, CD14+ monocytes were isolated fromFicoll-purified human peripheral blood mononuclear cells (PBMCs) usingRosetteSep human monocyte enrichment cocktail (Stemcell). The percentageof monocytes present after the enrichment was determined by flowcytometry on the FACSVerse (BD Biosciences) based on CD14 staining usingan APC-Cy7-conjugated mouse-anti-human CD14 detection antibody (BDBiosciences). Subsequently, CD14+ enriched PBMCs were seeded in 96-wellround bottomed tissue culture plates and incubated for 40 minutes withFcR Blocking Reagent (Miltenyi Biotec) containing hSIRPα.40A antibody(20 μg/mL and dilutions thereof) in PBS/1% BSA at 4° C. Next, cells werewashed three times with PBS/1% BSA and incubated with an Alexa Fluor647-labeled goat-anti-mouse IgG (Invitrogen) detection antibody inPBS/1% BSA for 40 minutes at 4° C. After this labeling procedure, cellswere washed two times, resuspended in PBS/1% BSA containing 0.1 μg/mLDAPI (BioLegend), and analysed by flow cytometry on the FACSVerse (BDBiosciences). Data were processed and analysed with FlowJo V10 software(FlowJo, LLC).

FIGS. 14A and B shows that hSIRPα.40A binds to primary human CD14+enriched monocytes. EC50 values represent the concentration at which 50%of the total binding signal is observed. To assess the blocking abilityof hSIRPα.40A, CD14+ enriched monocytes cells were seeded in 96-wellround bottomed tissue culture plates and incubated for 45 minutes withFcR Blocking Reagent (Miltenyi Biotec) and hSIRPα.40A antibody (20 μg/mLand dilutions thereof) in PBS/1% BSA at 4° C. Thereafter, cells werewashed three times with PBS/1% BSA and incubated with 10 μg/mL DyLight488-labeled recombinant hCD47/Fc-protein for 45 minutes at 4° C. Afterthis labeling procedure, cells were washed two times, resuspended inPBS/1% BSA containing 0.1 μg/mL DAPI (BioLegend), and analysed by flowcytometry on the FACSVerse (BD Biosciences). Data were processed andanalysed with FlowJo V10 software (FlowJo, LLC). FIGS. 14C and Ddemonstrates the ability of antibody hSIRPα.40A to block thehSIRPα/hCD47 interaction. IC50 values for the blockade of hCD47 werecalculated from this data. IC50 values represent the concentration atwhich half of the inhibition is observed.

Example 14: Functionality of hSIRPα.40A mAb in the Human GranulocytePhagocytosis Assay

To confirm the functionality of hSIRPα.40A in primary immune cells,granulocytes (e.g. effector cells) were isolated from healthy humandonor EDTA blood. First, the EDTA blood of each donor was pooled andcentrifuged at 300 g for 6 minutes at 20° C. Next, plasma was removed byaspiration, and the remaining blood cells were gently resuspended. Cellswere recovered in red blood cell (RBC) lysis buffer (155 mM NH4Cl; 10 mMKHCO3) and incubated for 10 minutes on ice. Next, cells were centrifugedat 300 g for 7 minutes. Supernatants containing lysed RBCs were removedby aspiration, and the remaining blood cells were gently resuspended inRBC lysis buffer and kept on ice for 1 minute. RBC lysis was neutralizedby adding assay medium (IMDM (Gibco) supplemented with 10% Fetal BovineSerum (Gibco) and Pen/Strep (Gibco)). Blood cells were centrifuged at300 g for 6 minutes and supernatants were removed by aspiration toremove remaining RBCs as much as possible. Subsequently,erythrocyte-lysed blood cells were resuspended in assay mediumcontaining 10 ng/mL IFNγ and cells were incubated for 1 hour at 37° C.,5% CO₂ and 95% humidity. Non-adherent blood cells containing humangranulocytes were collected by mild washing of the tissue culture platewith assay medium (monocytes are depleted due to adherence to theplastic surface). The percentage of granulocytes present in the cellsuspension was determined by flow cytometry on the FACSCanto II (BDBiosciences) based on high forward scatter (FSC) and side scatter (SSC).Binding of hSIRPα.40A to human granulocytes was assessed by incubatingthe cells for 30 minutes at 4° C. with hSIRPα.40A antibody (25 μg/mL anddilutions thereof) in PBS/1% BSA containing 10% autologous serum (PBS/1%BSA/10% serum). Next, cells were washed three times with PBS/1% BSA/10%serum and incubated for 30 minutes at 4° C. with a FITC-labeledgoat-anti-mouse Ig (BD Biosciences) detection antibody. After thislabeling procedure, cells were washed two times, resuspended in PBS/1%BSA/10% serum and analysed by flow cytometry on the FACSCanto II (BDBiosciences). Data were processed and analysed with FlowJo V10 software(FlowJo, LLC). FIG. 15A and the following Table 19 shows that hSIRPα.40Abinds to primary human granulocytes. EC₅₀ values represent theconcentration at which 50% of the total binding signal is observed.

TABLE 19 Donor 1 Antibody EC50 (nM) hSIRPα.40A 1.227 hSIRPα.50A 4.298

Next, Ramos (ECACC 85030802) target cells were fluorescently labeledwith cell proliferation dye eFluor450 (eBioscience). Labeling wasperformed according to manufacturer's instructions. Labeled target cellswere co-cultured for 2-3 hours at 37° C., 5% CO₂ and 95% humidity withisolated primary human granulocytes in a 1:1 ratio (7.5*10⁴ cells ofeach target and effector per well of a 96-well round bottomed tissueculture plate) in the presence of 0.1 μg/mL rituximab (anti-hCD20). Inaddition, cells were co-cultured with 0.1 μg/mL rituximab in presence of10 μg/mL hSIRPα.40A. Phagocytosis was assayed by determining thepercentage of granulocytes positive for eFluor450 using flow cytometryon the FACSCanto II (BD Biosciences). Data were processed and analysedwith FlowJo V10 software (FlowJo, LLC).

Compared to the mouse IgG1 isotype control, hSIRPα.40A potently enhancestumor cell phagocytosis induced by rituximab (FIG. 15B).

Example 15: Functionality of hSIRPα.40A mAb in the Human MacrophagePhagocytosis Assay

Blockade of CD47 by hSIRPα.40A enhances the phagocytosis of humanlymphoma cells tumor cells by human macrophages. Human macrophages weregenerated by first enriching CD14+ monocytes from Ficoll-purified humanperipheral blood mononuclear cells (PBMCs) using RosetteSep humanmonocyte enrichment cocktail (Stemcell). Monocytes were seeded intoCellCarrier 96-well flat-bottom microplates (Perkin Elmer) and culturedin macrophage medium (IMDM (Gibco) supplemented with 8.5% Fetal BovineSerum (Gibco) and Pen/Strep (Gibco)) containing 50 ng/mL human monocytecolony stimulating factor (M-CSF) for 7 days at 37° C., 5% CO₂ and 95%humidity to promote differentiation into macrophages. Thesemonocyte-derived macrophages (MDMs) become adherent allowing other cellsto be washed away. Human Raji lymphoma cells were counted and labeledwith cell proliferation dye eFluor450 (eBioscience) followingmanufacturer's instructions. After labeling, the lymphoma cells weremixed with assay medium (RPMI (Gibco) supplemented with 10% Fetal BovineSerum (Gibco) and Pen/Strep (Gibco)) containing 100 μg/mL anti-hSIRPαantibodies and dilutions thereof, the respective isotype controlantibody, and 1μg/mL rituximab (anti-hCD20). The lymphoma cells werethen added to the individual wells containing MDMs at a ratio of 2.5:1tumor cells per phagocyte, mixed and incubated at 37° C., 5% CO₂ and 95%humidity for 2 hours. After the incubation, the wells were washed withPBS to remove most of the non-phagocytosed tumor cells, and cells werefixed with 2% formaldehyde for 10 min at RT. The wells were then washedand maintained in PBS/3% BSA in dark at 4° C. overnight. Lymphoma cellspresent in the wells were stained with biotin-conjugated anti-human CD19clone HIB19 (eBioscience) for 1 hour at RT, and subsequently werecounterstained with Alexa Fluor 488-conjugated streptavidin (ThermoFisher Scientific) for 1 hour at RT. Next, nuclei were stained withDRAQS (Thermo Fisher Scientific) for 10 minutes at RT, mixture wasremoved, and PBS was added to each well. Cells were analysed with theOperetta automated fluorescence microscope (Perkin Elmer). Data wereprocessed and analysed with Columbus V2.6 software.

As shown in FIG. 16, hSIRPα.40A enhances rituximab-mediated phagocytosisactivity. The phagocytosis of human lymphoma cells was quantified usinga phagocytosis index, as follows: (number of tumor cells insidemacrophages/number of macrophages)*100; counting at least 200macrophages per sample.

Example 16: Humanized Antibody Design and CDR Grafting

The mouse hSIRPα.40A antibody was humanized using CDR-graftingtechnology (see e.g. U.S. Pat. No. 5,225,539 and Williams, D.G. et al.,2010, Antibody Engineering, volume 1, Chapter 21). First, human germlinesequences were identified using IgBLAST (Ye J. et al., Nucleic AcidsRes. 41:W34-40 (2013). For the hSIRPα.40A VH human germline sequence,V-gene IGHV1-46*01 was identified (62.2% identity) and for the VL humangermline sequence IGKV1-39*01 was identified (68.4% identity). These twogermline sequences were used as template to graft the mouse CDRs,resulting in the following two cDNA constructs: SEQ ID NO: 87 (VH) andSEQ ID NO: 99 (VL).

Next, a database was constructed containing all human sequencesavailable in the IMGT database (Lefranc, M.-P. et al., Nucleic Acid Res.27:209-212 (1999)) identifying 85,848 individual sequences. Thesesequences were queried using TBLASTN (2.2.31+) to identify templatesequences that demonstrated the highest identify to the framework ofhSIRPα.40A VH and VL sequences. Four VH and four VL sequences wereidentified that demonstrated a similarity score of 80% or higher andthat displayed similar CDR lengths, preferably identical to those inhSIRPα.40A VH CDR1, CDR2, CDR3 and VL CDR1, CDR2 and CDR3, respectively.

For the heavy chain, the frameworks encoded by GenBank (Benson, D.A. etal., Nucleic Acids Res. 41(D1):D36-42 (2013)) accession # L39130,DJ031925, DJ326840, and EF177968 were selected as templates for graftingof the hSIRPα.40A VH CDRs, resulting in the following cDNA constructs:SEQ ID NO: 77, 79, 81 and 83, respectively. For the light chain, theframeworks encoded by GenBank accession #AY731031, DQ840993, AY942002and DQ535171 were selected as templates for straight grafting of thehSIRPα.40A VL CDRs, resulting in the following cDNA constructs: SEQ IDNO: 89, 91, 93 and 95. Additionally, a database was constructedcontaining all humanized antibody sequences available in the publicdomain, identifying 300 sequences. These sequences were queried usingBLASTP (2.2.31+) to identify template sequences that demonstrated thehighest identify to the framework of hSIRPα.40A VH and VL sequences. Forthe heavy chain, the framework of Gemtuzumab was selected as template,for grafting of the hSIRPα.40A VH CDRs, resulting in the following cDNAconstruct: SEQ ID NO: 85. For the light chain, the framework ofAlacizumab was selected as template, for grafting of the hSIRPα.40A VLCDRs, resulting in the following cDNA construct: SEQ ID NO: 97

Framework and CDR definition were those as described by Kabat et al.(“Sequences of Proteins of Immunological Interest”, Kabat, E., et al.,US Department of Health and Human Services, (1983)).

To study the effect of humanized framework residues on the structure ofthe Fv, a homology model of the mouse hSIRPα.40A Fv was made using the‘Antibody Modeling Cascade’ (default parameters) within Discovery Studio4.5. The homology model was built on basis of PDB ID 3UMT, for the lightchain, PDB ID 1EHL for the heavy chain, and PDB ID 3BGF for the Fv. TheCDRs were grafted in silico to study residues that are close to any ofthe CDRs and which might affect the loop conformation, referred to asVernier residues. Residues that might affect the loop conformation, andwhich are within <5 Å A to the CDR surface were identified andsubstituted with the mouse amino acid at this position. The resultingtemplates were checked for the presence of post translationalmodification (PTM) motifs using Discovery Studio 4.5 and where possible(i.e. non-CDR, non-Vernier residues) changed to prevent a PTM. The VHCDR2 contained a glycosylation site that was removed by an aspargine toserine mutation.

CDRs were grafted on each of the identified templates, expressed as ahuman IgG2 (SEQ ID NO: 68), kappa (SEQ ID NO: 64) antibody cloned in thepcDNA3.1(+) vector (Thermo Fisher Scientific) and for transienttransfection in FreeStyle 293-F human embryonic kidney cells(HEK293T/17, ATCC CRL-11268).

Example 17: Synthesis, Expression and Purification of Chimeric andHumanized Constructs

Plasmids encoding the heavy chain and light chain humanized constructswere mixed in a 1:1 ratio (30 μg in total) and transiently expressed bytransfection into FreeStyle 293-F cells using 293fectin transfectionreagent (Invitrogen) following the manufacturer's instructions.Supernatants (30 ml) were harvested after 7 days, filtered over a 0.22μm filter, and antibodies were purified using MabSelect Sure Protein Aresin according to the manufacturer's instructions (GE Healthcare).Buffer was exchanged for 10 mM Histidine, 100 mM NaCl pH 5.5 bufferusing Zeba desalting columns (Thermo Fisher Scientific). Theconcentration of purified antibodies was determined based on OD280(Nanodrop ND-1000). Endotoxin level was determined by LAL-test accordingto the manufacturer's instructions (Lonza).

Example 18: Binding of Humanized SIRPα Antibodies

Binding of the parental and humanized antibodies to hSIRPα was assessedby flow cytometry using the CHO-K1.hSIRPαV1 stable cell line.CHO-K1.hSIRPαV1 cells were seeded in 96-well round bottomed tissueculture plates and incubated for 40 minutes with the humanizedhSIRPα.40A antibody variants (20 μg/mL and dilutions thereof) in PBS/1%BSA at 4° C. Next, cells were washed three times with PBS/1% BSA andincubated for 40 minutes at 4° C. with either an Alexa Fluor 647-labeledgoat-anti-mouse IgG (Invitrogen), or an Alexa Fluor 647-labeleddonkey-anti-human IgG (Jackson Immuno Research) detection antibody inPBS/1% BSA. After this labeling procedure, cells were washed two times,resuspended in PBS/1% BSA, containing 0.1 μg/mL DAPI (BioLegend), andanalysed by flow cytometry on the FACSVerse (BD Biosciences). Data wereprocessed and analysed with FlowJo V10 software (FlowJo, LLC). EC50values, the concentration at which 50% of the total binding signal isobserved, were calculated using GraphPad Prism 6 (GraphPad Software,Inc.) (FIG. 17 and Table 20).

TABLE 20 hSIRPαV1 Antibody EC50 (nM) hSIRPα.40A 0.022 hSIRPα.40H1L1 ndhSIRPα.40H1L2 nd hSIRPα.40H1L3 nd hSIRPα.40H1L4 nd hSIRPα.40H1L5 ndhSIRPα.40H1L6 nd hSIRPα.40H2L1 0.264 hSIRPα.40H2L2 0.298 hSIRPα.40H2L30.300 hSIRPα.40H2L4 0.315 hSIRPα.40H2L5 0.284 hSIRPα.40H2L6 0.251hSIRPα.40H3L1 1.644 hSIRPα.40H3L2 1.404 hSIRPα.40H3L3 1.501hSIRPα.40H3L4 0.693 hSIRPα.40H3L5 2.302 hSIRPα.40H3L6 0.833hSIRPα.40H4L1 3.308 hSIRPα.40H4L2 3.360 hSIRPα.40H4L3 3.072hSIRPα.40H4L4 3.471 hSIRPα.40H4L5 4.828 hSIRPα.40H4L6 3.028hSIRPα.40H5L1 2.011 hSIRPα.40H5L2 1.919 hSIRPα.40H5L3 2.268hSIRPα.40H5L4 0.869 hSIRPα.40H5L5 2.954 hSIRPα.40H5L6 2.197hSIRPα.40H6L1 2.349 hSIRPα.40H6L2 3.002 hSIRPα.40H6L3 3.014hSIRPα.40H6L4 1.279 hSIRPα.40H6L5 3.785 hSIRPα.40H6L6 2.677 nd, notdetected

Example 19: Blockade of hCD47 Binding to hSIRPα by Humanized hSIRPα.40AAntibodies

hCD47 blockade was assessed by flow cytometry for the full panel ofhumanized hSIRPα.40A antibodies. To this end, the U-937 (ATCCCRL-1593.2) monocyte cell line was used as the source of hSIRPα in theassay. U-937 cells were seeded in 96-well round bottomed tissue cultureplates and incubated for 45 minutes with FcR Blocking Reagent (MiltenyiBiotec) and the parental or humanized hSIRPα.40A antibody variants (20μg/mL and dilutions thereof) in PBS/1% BSA at 4° C. Next, cells werewashed three times with PBS/1% BSA and incubated with 10 μg/mL DyLight488-labeled recombinant hCD47/Fc-protein for 30 minutes at 4° C. Afterthis labeling procedure, cells were washed two times, resuspended inPBS/1% BSA containing 0.1 μg/mL DAPI (BioLegend), and analysed by flowcytometry on the FACSVerse (BD Biosciences). Data were processed andanalysed with FlowJo V10 software (FlowJo, LLC) and plotted usingGraphPad Prism 6 (GraphPad Software, Inc.).

As depicted in FIG. 18 and the following Table 21, binding ofrecombinant hCD47 fused to an Fc domain of human IgG1 was monitored inthe presence of increasing amounts of the humanized hSIRPα.40A antibodyvariants. Humanized hSIRPα.40A blocked the hSIRPα/hCD47 interaction,using the flow cytometry-based method described above. IC50 values forthe blockade of hCD47 were calculated from this data. IC50 valuesrepresent the concentration at which half of the inhibition is observed.

TABLE 21 U-937 Antibody IC50 (nM) hSIRPα.40A 1.122 hSIRPα.40H1L1 ndhSIRPα.40H1L2 nd hSIRPα.40H1L3 nd hSIRPα.40H1L4 nd hSIRPα.40H1L5 ndhSIRPα.40H1L6 nd hSIRPα.40H2L1 0.638 hSIRPα.40H2L2 0.773 hSIRPα.40H2L30.685 hSIRPα.40H2L4 0.718 hSIRPα.40H2L5 0.745 hSIRPα.40H2L6 0.901hSIRPα.40H3L1 0.980* hSIRPα.40H3L2 nd h5IRPα.40H3L3 2.625* hSIRPα.40H3L41.784* hSIRPα.40H3L5 2.435* hSIRPα.40H3L6 97.762* hSIRPα.40H4L1 10.002*hSIRPα.40H4L2 7.579* hSIRPα.40H4L3 75.422* hSIRPα.40H4L4 3.153*hSIRPα.40H4L5 5.171* hSIRPα.40H4L6 3.512* hSIRPα.40H5L1 34.977*hSIRPα.40H5L2 nd hSIRPα.40H5L3 nd hSIRPα.40H5L4 10.772* hSIRPα.40H5L5 ndhSIRPα.40H5L6 0.247* hSIRPα.40H6L1 2.391* hSIRPα.40H6L2 20.427*hSIRPα.40H6L3 9.208* hSIRPα.40H6L4 3.797* hSIRPα.40H6L5 20.421*hSIRPα.40H6L6 9.750* Values indicated with * were extrapolated; nd, notdetected

Example 20: Binding Domain of hSIRPα.40A

To identify the binding region of hSIRPα.40A, several SIRPβ1exchange-mutants were designed based on the human SIRPβ1 and SIRPγ aminoacid sequences. Based on the fold of SIRPα/β1/γ, the extracellularregion can be subdivided into three separate domains: the Ig-like(immunoglobulin-like) V-type (IgV), Ig-like C1-type (IgC1), and Ig-likeC2-type (IgC2) domain. The IgV domain is also known as theligand-binding N-terminal domain of SIRPα and SIRPγ (which binds toCD47). The human SIRPβ1/γ mutants were designed based on the full lengthhSIRPβ1 sequence (SEQ ID NO: 38) and each individual Ig-like domain wassubstituted for the equivalent domain of human SIRPγ (SEQ ID NO: 40).The cDNAs encoding the constructs, hSIRP-VγC1βC2β (SEQ ID NO: 110),hSIRP-VβC1γC2β (SEQ ID NO: 112), and hSIRP-VβC1βC2γ (SEQ ID NO: 114)were synthesized (GeneArt) and subcloned into the pCI-neo vector.Binding of hSIRPα.40A to the exchange mutants was tested using CELIS A.To this end, CHO-K1 cells were transiently transfected, usingLipofectamine 2000, with the pCI-neo vectors encoding hSIRPαV1,hSIRPαV2, hSIRPβ1, hSIRP-VγC1βC2β, hSIRP-VβC1γC2β, and hSIRP-VβC1βC2γ,respectively. The transfected cells were cultured at 37° C., 5% CO₂ and95% humidity in medium (DMEM-F12 (Gibco) with 5% New Born Calf serum(Biowest) and Pen/Strep (Gibco)) until confluent. Subsequently, cellswere trypsinized and seeded in 96-well flat-bottom tissue culture platesand cultured at 37° C., 5% CO₂ and 95% humidity in culture medium untilconfluent. Then, culture medium was removed and cells were incubated for1 hour at 37° C., 5% CO₂ and 95% humidity with hSIRPα.40A, hSIRPα.50A,and anti-hSIRPγ clone SE5A5 antibodies. Next, cells were washed withPBS-T and incubated for 1 hour at 37° C., 5% CO₂ and 95% humidity withgoat-anti-mouse IgG-HRP conjugate (Southern Biotech). After that, cellswere washed three times with PBS-T and anti-hSIRPγ immunoreactivity wasvisualized with TMB Stabilized Chromogen (Invitrogen). Reactions werestopped with 0.5 M H₂SO₄ and absorbances were read at 450 and 610 nm.

The antibody of the present invention demonstrated gain of binding tothe hSIRP-VγC1βC2β mutant, indicating that hSIRPα.40A binds to the IgVdomain of hSIRPγ and hSIRPγ (FIG. 19 and Table 22). EC50 valuesrepresent the concentration at which 50% of the total binding signal isobserved (average and SD were calculated from values of two independentexperiments).

TABLE 22 Antibody anti-hSIRPα hSIRPα.40A hSIRPα.50A (clone SE5A5)hSIRPαV1 EC50(nM) 0.133 0.968 0.350 SD 0.065 0.432 0.136 hSIRPαV2EC50(nM) 0.101 0.821 0.224 SD 0.051 0.183 0.076 hSIRPβ1 EC50(nM) nd nd0.249 SD nd nd 0.091 hSIRP-VγClβC2β EC50(nM) 0.123 2.524 0.287 SD 0.0400.609 0.026 hSIRP-VβC1γC2β EC50(nM) nd nd 0.309 SD nd nd 0.140hSIRP-VβC1βC2γ EC50(nM) nd nd 0.231 SD nd nd 0.079 nd, not detected

To pinpoint the amino acids for interaction of hSIRPα.40A with the IgVdomain, several point mutants of hSIRPαV1 were generated based on singleamino acid differences between hSIRPαV1/V2 and hSIRPβ1. The followingsequence alignment shows an alignment of the hSIRPα and hSIRPβ1 IgVdomain.

Sequence Alignment of the IgV Domain:

Amino acids in the hSIRPα IgV domain that are altered in hSIRPβ1 weremutated by using the QuikChange II Site-Directed Mutagenesis Kit(Stratagene) and the full length hSIRPαV1 sequence (SEQ ID NO: 33) asdonor cDNA. Binding of hSIRPα.40A to hSIRPαV1 point mutants was testedusing CELISA. To this end, CHO-K1 cells were transiently transfected,using Lipofectamine 2000, with cDNA encoding the full length openreading frame of hSIRPαV1 and mutants thereof, and hSIRPβ1 subclonedinto the pCI-neo vector. Transfected cells were seeded in culture medium(DMEM-F12 (Gibco) supplemented with 5% New Born Calf Serum (BioWest) andPen/Strep (Gibco)) in 96-well flat-bottom tissue culture plates andincubated at 37° C., 5% CO₂ and 95% humidity for 24 hours. Subsequently,culture medium was removed and cells were incubated for 1 hour at 37°C., 5% CO₂ and 95% humidity with purified hSIRPα antibodies (used at 10μg/mL and dilutions thereof). Next, cells were washed with PBS-T andincubated for 1 hour at 37° C., 5% CO₂ and 95% humidity withgoat-anti-mouse IgG-HRP (Southern Biotech). Subsequently, cells werewashed three times with PBS-T and immunoreactivity against hSIRPαV1,hSIRPαV1 mutants, and hSIRPβ1 was visualized with TMB StabilizedChromogen (Invitrogen). Reactions were stopped with 0.5 M H₂SO₄ andabsorbances were read at 450 and 610 nm. EC₅₀ values, the concentrationat which 50% of the total binding signal is observed, were calculatedusing GraphPad Prism 6 (GraphPad Software, Inc.) (average and SD werecalculated from values of two independent experiments).

As shown in FIG. 20 and the following Table 23, the Proline at position74 (P74) constitues a crucial amino acid for the specific binding ofhSIRPα.40A to hSIRPαV1. Expression of hSIRPαV1(P74A) (SEQ ID NO: 61),where P74 is converted to Alanine, on CHO-K1 cells results in loss ofhSIRPα.40A antibody binding. This proline is not present in the IgVdomain sequence of hSIRPβ1, and could play a role in the correctconformation of the IgV domain.

TABLE 23 hSIRPαV1 binding hSIRPβ1 binding hSIRPαV1(P74A) binding EC50(nM) EC50 (nM) EC50 (nM) Antibody Average SD Average SD Average SDhSIRPα.40A 0.065 0.006 nd nd nd nd hSIRPα.50A 0.534 0.152 nd nd nd ndanti-hSIRPα (clone SE5A5) 0.163 0.008 0.156 0.009 0.149 0.013 nd, notdetected

Example 21: Functionality of Chimeric hSIRPα.40A mAb Variants in theHuman Macrophage Phagocytosis Assay

The functionality of hSIRPα.40A variable domains, grafted on differentFc constant domains, was assessed by an in vitro phagocytosis assayusing human macrophages. Experimental conditions for the humanmacrophage phagocytosis assay were similar as explained in Example 15above. Labelled Raji lymphoma cells were mixed with assay mediumcontaining either 10 μg/mL or 1μg/mL chimeric hSIRPα.40A antibodyvariants and 1μg/mL rituximab and then added to MDMs at a ratio of 2.5:1tumor cells per phagocyte. Cells were incubated at 37° C., 5% CO₂ and95% humidity for 2 hours.

Analysis was performed with the Operetta automated fluorescencemicroscope (Perkin Elmer) and data were processed and analysed withColumbus V2.6 software. The phagocytosis of human lymphoma cells wasquantified using a phagocytosis index, as follows: (number of tumorcells inside macrophages/number of macrophages)*100; counting at least200 macrophages per sample.

As shown in FIG. 21, the wild-type (WT) chimeric hSIRPα.40A.hIgG4antibody does not enhance rituximab-mediated phagocytosis, whereas inertchimeric hSIRPα.40A.hIgG1 (SEQ ID NO: 119) antibody variants containingN297Q (SEQ ID NO: 126), L234A.L235A (LALA) (SEQ ID NO: 123), orL234A.L235A.P329G (LALAPG) (SEQ ID NO: 125) mutations enhancerituximab-mediated phagocytosis activity in a concentration-dependentmanner. Likewise, hSIRPα.40A.hIgG2 and the inert chimerichSIRPα.40A.hIgG2 antibody variant containingV234A.G237A.P238S.H268A.V309L.A330S.P331S (Sigma) (SEQ ID NO: 122)mutations enhance rituximab-mediated phagocytosis activity in aconcentration-dependent manner.

Example 22: Functionality of Humanized hSIRPα.40A mAb Variants in theHuman Macrophage Phagocytosis Assay

The functionality of a selected set of the humanized hSIRPα.40A antibodyvariants was assessed by an in vitro phagocytosis assay using humanmacrophages. Experimental conditions for the human macrophagephagocytosis assay were similar as explained in Example 6.

As shown in FIG. 22, the humanized hSIRPα.40A antibody variants enhancerituximab-mediated phagocytosis activity in a concentration-dependentmanner similar to antibody KWAR23 grafted on a hIgG2 Fc.

Example 23: Functionality of Chimeric hSIRPα.50A mAb Variants in theHuman Macrophage Phagocytosis Assay

The functionality of hSIRPα.50A variable domains, grafted on differentFc constant domains, was assessed by in vitro phagocytosis assays usinghuman macrophages. As shown in FIG. 23A, the chimeric hSIRPα.50A.hIgG4antibody marginally enhances rituximab-mediated phagocytosis, whereasthe chimeric hSIRPα.50A.hIgG2 antibody enhances rituximab-mediatedphagocytosis activity similar to the murine hSIRPα.50A.mIgG1 (SEQ ID NO:120) antibody. FIG. 23B demonstrates that the chimeric hSIRPα.50A.hIgG2antibody potently enhances tumor cell phagocytosis induced by rituximabin a concentration-dependent manner as compared to the human IgG2isotype control. Similarly, hSIRPα.50A.hIgG2 enhanceddaratumumab-mediated phagocytosis (anti-hCD38, used at 0.05 μg/mL) (FIG.23C).

In addition, hSIRPα.50A.hIgG2 also enhanced rituximab-mediatedphagocytosis in human granulocytes. As shown in FIG. 23D, the chimerichSIRPα.50A.hIgG2 antibody enhances phagocytosis activity induced byrituximab to a similar extend as the murine hSIRPα.50A.mIgG1 antibody.Likewise, as shown in FIG. 24A, the chimeric hSIRPα.50A.hIgG1.N297Q,hSIRPα.50A.hIgG4.N297Q (SEQ ID NO: 127) or hSIRPα.50A.hIgG2 antibodiesenhance rituximab-mediated phagocytosis activity by human MDMs to asimilar extent as the murine hSIRPα.50A.mIgG1 antibody (rituximab usedat 1 μg/mL). Similar observations were made in FIG. 24B whenphagocytosis was induced by daratumumab (0.05 μg/mL). As shown in FIG.25, the chimeric hSIRPα.50A.hIgGl.N297Q and hSIRPα.50AhIgG1.L234A.L235A.P329G antibodies also enhance rituximab-mediatedphagocytosis activity by human MDMs to a similar extent as the orhSIRPα.50A.hIgG2 antibody (rituximab used at 1 μg/mL). Chimeric variantsof hSIRPα.50A mAb containing a wild-type hIgG1 or hIgG4 Fc region didnot enhance tumor cell phagocytosis.

Example 24: Comparison of KWAR23, clone 18D5, hSIRPα.50A, and hSIRPα.40AAntibodies

A direct comparison of the specificity of monoclonal anti-hSIRPαantibodies KWAR23, clone 18D5 (SEQ ID NO: 128; SEQ ID NO: 129) fromW02017/178653, hSIRPα.50A, and hSIRPα.40A for binding to hSIRPαV1,hSIRPαV1(P74A), hSIRPαV2, and hSIRPβ1 was evaluated by CELISA.Reactivity was confirmed using CHO-K1 cells (ATCC CCL-61) expressing acDNA encoding the full length open reading frame of hSIRPαV1,hSIRPαV1(P74A), hSIRPαV2, and hSIRPβ1 subcloned into the pCI-neo vector(Promega, Madison, Wis.). CHO-K1.hSIRPαV1, CHO-K1.hSIRPαV1(P74A),CHO-K1.hSIRPαV2, and CHO-K1.hSIRPβ1 cells were seeded in culture medium(DMEM-F12 (Gibco) supplemented with 5% New Born Calf Serum (BioWest) andPen/Strep (Gibco)) in 96-well flat-bottom tissue culture plates andincubated at 37° C., 5% CO₂ and 95% humidity for 24 hours. Subsequently,culture medium was removed and cells were incubated for 1 hour at 37°C., 5% CO₂ and 95% humidity with purified hSIRPα antibodies (used at 10μg/mL and dilutions thereof). Next, cells were washed with PBS-T andincubated for 1 hour at 37° C., 5% CO₂ and 95% humidity withgoat-anti-mouse IgG-HRP (Southern Biotech). Subsequently, cells werewashed three times with PBS-T and immunoreactivity against hSIRPαV1,hSIRPαV1(P74A), hSIRPαV2, and hSIRPβ1 was visualized with TMB StabilizedChromogen (Invitrogen). Reactions were stopped with 0.5 M H₂SO₄ andabsorbances were read at 450 and 610 nm. EC50 values, the concentrationat which 50% of the total binding signal is observed, were calculatedusing GraphPad Prism 6 (GraphPad Software, Inc.).

Binding to hSIRPγ was assessed by flow cytometry using the Jurkat E6.1 Tcell leukemia cell line (ECACC 88042803). Jurkat cells were seeded in96-well round bottomed tissue culture plates and incubated for 40minutes with the anti-hSIRPα antibodies (20 μg/mL and dilutions thereof)in PBS/1% BSA at 4° C. Next, cells were washed three times with PBS/1%BSA and incubated for 40 minutes at 4° C. with an Alexa Fluor647-labeled goat-anti-mouse IgG (Invitrogen) detection antibody inPBS/1% BSA. After this labeling procedure, cells were washed two times,resuspended in PBS/1% BSA, containing 0.1 μg/mL DAPI (BioLegend), andanalysed by flow cytometry on the FACSVerse (BD Biosciences). Data wereprocessed and analysed with FlowJo V10 software (FlowJo, LLC). EC₅₀values, the concentration at which 50% of the total binding signal isobserved, were calculated using GraphPad Prism 6 (GraphPad Software,Inc.).

As depicted in Table 24, KWAR23 and clone 18D5 antibodies cross-reactwith at least hSIRPβ1 and the P74A variant of hSIRPαV1. The hSIRPα.50A,and hSIRPα.40A antibodies of the present invention do not bind to eitherhSIRPβ1 or the P74A variant of hSIRPαV1 under the tested conditions. Inthis regard, the hSIRPα.50A, and hSIRPα.40A antibodies of the presentinvention similarly distinguish from antibody clone SIRP29 fromWO2013/056352. FIGS. 7A and B of WO2017/178653 compares clone SIRP29 andKWAR23 binding to SIRPβ1 (referred to as “sirp-b”, Product No.ABIN3077231 from antibodies-online.com), demonstrating that each ofclone SIRP29 and KWAR23 has nanomolar affinity for SIRPβ1.

TABLE 24 hSIRPαV1(P74A) hSIRPαV2 hSIRPβ1 hSIRPαV1 binding bindingbinding binding hSIRPγ binding Antibody EC50 (nM) EC50 (nM) EC50 (nM)EC50 (nM) EC50 (nM) hSIRPα.40A 0.114 nd 0.093 nd 0.369 hSIRPα.50A 0.773nd 0.645 nd — KWAR23 0.070 0.049 0.049 0.033 0.003 18D5 0.134 0.055 nd0.055 nd nd, not detected; -, not tested

hCD47 blockade for the KWAR23, clone 18D5, and hSIRPα.40A antibodies wasassessed by flow cytometry. For this purpose, THP-1 (ATCC TIB-202) andU-937 (ATCC CRL-1593.2) monocyte cell lines were used as the source ofhSIRPα in the assay. THP-1 and U-937 cells were seeded in 96-well roundbottomed tissue culture plates and incubated for 45 minutes with FcRBlocking Reagent (Miltenyi Biotec) and indicated anti-hSIRPα antibodies(20 μg/mL and dilutions thereof) in PBS/1% BSA at 4° C. Next, cells werewashed three times with PBS/1% BSA and incubated with 10 μg/mL DyLight488-labeled recombinant hCD47/Fc-protein for 30 minutes at 4° C. Afterthis labeling procedure, cells were washed two times, resuspended inPBS/1% BSA containing 0.1 μg/mL DAPI (BioLegend), and analysed by flowcytometry on the FACSVerse (BD Biosciences). Data were processed andanalysed with FlowJo V10 software (FlowJo, LLC) and plotted usingGraphPad Prism 6 (GraphPad Software, Inc.). Binding of recombinant hCD47fused to an Fc domain of human IgG1 was monitored in the presence ofincreasing amounts of the anti-hSIRPα antibodies. IC50 values for theblockade of hCD47 were calculated from this data. IC50 values representthe concentration at which half of the inhibition is observed.

As depicted in Table 18 and Table 25, hSIRPα.40A, hSIRPα.50A, and KWAR23antibodies block rhCD47/Fc binding to both the THP-1 and U-937 monocytecell lines which express the hSIRPαV2 and hSIRPαV1 allele, respectively.Antibody clone 18D5 blocks rhCD47/Fc binding to the U-937 monocyte cellline but does not block rhCD47/Fc binding to the THP-1 monocyte cellline, in line with the observation that 18D5 does not bind to hSIRPαV2(Table 24). In this regard, the hSIRPα.50A, and hSIRPα.40A antibodies ofthe present invention similarly distinguish from antibody clone 18D5.

TABLE 25 THP-1 U-937 Antibody IC50 (nM) IC50 (nM) hSIRPα.40A 0.548 1.417KWAR23 0.132 0.284 18D5 nd 1.522 nd, not detected

Example 25: Mapping the Interaction Interface between hSIRPα-hSIRPα.40Aand hSIRPα-hSIRPα.50A

The amino acids on hSIRPα that are bound by hSIRPα.40A or hSIRPα.50Awere elucidated by a procedure that involves deuterated chemicalcross-linking followed by enzymatic digestion and detection using massspectrometry. First, antibody hSIRPα.40A and antigen rhSIRPα-HIS(SinoBiological 11612-H08H-100, SEQ ID NO: 132), or antibody hSIRPα.50Aand antigen rhSIRPα-HIS were incubated to promote binding and integrityand aggregation level were verified by Ultraflex III MALDI TOF massspectrometer (Bruker) equipped with a HM4 interaction module (CovalX).For these control experiments a dilution series of 10 μL samples ofantibody or antigen (1- to 128-fold dilution, starting at 1 mg/mL) wereprepared. Of each sample 9 μL was submitted to cross-linking using K200MALDI MS analysis kit, according to the manufacturer's instructions(CovalX) and incubated for 180 minutes, while 1μL was directly used formass spectrometry analysis (High-Mass MALDI). The mass spectrometryanalysis showed the antibody and antigen had the expected molecularweight: hSIRPα.40A=151.68 kDa (152.78 kDa with cross-linker),hSIRPα.50A=151.80 kD (153.17 kDa with cross-linker), andrhSIRPα-HIS=46.05 kDa (48.67 kDa with cross-linker). Forcharacterization of the antigen-antibody complex, a mixture was madewith an excess of antigen (antigen:antibody ratio forrhSIRPα-HIS:hSIRPα.40A 10.8 μM:8.5 μM, and antigen:antibody ratio forrhSIRPα-HIS:hSIRPα.50A 5.4 μM:2.13 μM). A 9 μL sample of theantigen-antibody mixture was submitted to cross-linking using K200 MALDIMS analysis kit, according to the manufacturer's instructions, while 1μL was directly used for mass spectrometry analysis. The detected massof the antibody and antigen (hSIRPα.40A: 151.18 kDa, rhSIRPα-HIS 45.93kDa, hSIRPα.50A: 151.69 kDa, rhSIRPα-HIS 46.18 kDa) corresponds to themolecular weight as detected previously. The antigen-antibody complexes,after cross-linking, were detected as two non-covalent complexes with a1:1 (195.24 kDa) and 2:1 (240.48 kDa) stoichiometry forrhSIRPα-HIS:hSIRPα.40A, and as one non-covalent complex with a 1:1(198.24 kDa) stoichiometry for rhSIRPα-HIS:hSIRPα.50A. Antibody andantigen bound non-covalent; non-covalent aggregates or non-specificmultimers were not detected.

Next, peptide mass fingerprinting of rhSIRPα-HIS was performed. Sampleswere submitted to ASP-N, trypsin, chymotrypsin, elastase and thermolysin(Roche Diagnostic) proteolysis, following manufacturer's instructionsfollowed by analysis by nLC-LTQ Orbitrap MS/MS using an Ultimate 3000(Dionex) system in line with a LTQ Orbitrap XL mass spectrometer (ThermoScientific). This proteolysis array resulted in 98% of the sequencebeing covered by the identified peptides.

To determine the interacting amino acids of antibody hSIRPα.40A andhSIRPα.50A on rhSIRPα-HIS antigen with high resolution, theantigen-antibody complex (rhSIRPα-HIS:hSIRPα.40A ratio 10.8 μM:8.5 μM,rhSIRPα-HIS:hSIRPα.50A ratio 5.4 p.1\4:2.13 μM) was incubated withdeuterated cross-linkers d0/d12 (K200 MALDI Kit) for 180 minutes andsubjected to multi-enzymatic cleavage with the enzymes ASP-N, trypsin,chymotrypsin, elastase and thermolysin. After enrichment of thecross-linked peptides, the samples were analyzed by high-resolution massspectrometry (nLC-Orbitrap MS) and the data generated were analyzedusing XQuest (Jin Lee, Mol. Biosyst. 4:816-823 (2008)) and Stavrox(Gotze et al., J. Am. Soc. Mass Spectrom. 23:76-87 (2012)). Theinteracting amino acids of hSIRPα.40A and hSIRPα.50A to rhSIRPα-HIS weremapped onto human SIRPαV1 (SEQ ID NO: 34). Cross-linked residues ofhSIRPα.40A are depicted as bold, boxed, and hSIRPα.50A as bold,underlined:

MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQ K EG H FPRVTTVSDLTKRN NMDF

IRIGNITPADAGT YY CVKFRKGSPDDVEF

SGAGTELSVRA

PSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYA SVQVPRK

The C-alpha distance between residue P74 and the identified cross-linkedresidues was measured in Discovery Studio using the crystal structure ofSIRPα (PDB ID 4CMM). The cross-linked residues identified for hSIRPα.50Aare within 14.0 to 21.4 angstrom C-alpha atom distance from residue P74;the cross-linked residues identified for hSIRPα.40A are within 16.2 to33.5 angstrom C-alpha atom distance from residue P74. The C-alphadistances fit within the expected range for an epitope-paratope surfacearea of 700 Å² (Rowley et al., Biotech. Ann. Rev. 10:151-188 (2004)).The identified residues and surface area are distinctly different fromthe binding epitope of anti-hSIRPα antibody KWAR23 (Ring et al., Proc.Natl Acad. Sci. USA 114:E10578-E10585 (2017)).

Example 26: Comparison of hSIRPα Antibodies for Binding to hSIRPαV1,hSIRPαV1(P74A), and hSIRPβ1

The specificity of monoclonal anti-hSIRPα antibodies (e.g., includingthe hSIRPα antibodies known in the art, KWAR23 (U.S. Patent CA2939293Al), 18D5 (Patent WO2017/178653 A2), and various commercially availablehSIRPα antibodies) for binding to hSIRPαV1, hSIRPαV1(P74A), and hSIRPβ1was evaluated by CELISA. Reactivity was confirmed using CHO-K1 cells(ATCC CCL-61) expressing a cDNA encoding the full length open readingframe of hSIRPαV1, hSIRPαV1(P74A), and hSIRPβ1 subcloned into thepCI-neo vector (Promega, Madison, Wis.). CHO-Kl.hSIRPαV1,CHO-K1.hSIRPαV1(P74A), and CHO-K1.hSIRPβ1 cells were seeded in culturemedium (DMEM-F12 (Gibco) supplemented with 5% New Born Calf Serum(BioWest) and Pen/Strep (Gibco)) in 96-well flat-bottom tissue cultureplates and incubated at 37° C., 5% CO₂ and 95% humidity for 24 hours.Subsequently, culture medium was removed and cells were incubated for 1hour at 37° C., 5% CO₂ and 95% humidity with purified hSIRPα antibodies(used at 10 μg/ml and dilutions thereof). Next, cells were washed withPBS-T and incubated for 1 hour at 37° C., 5% CO₂ and 95% humidity witheither goat-anti-mouse IgG-HRP (Southern Biotech), goat-anti-humanIgG-HRP (Jackson Immuno Research), or goat-anti-rabbit IgG-HRP (SouthernBiotech). Subsequently, cells were washed three times with PBS-T andimmunoreactivity against hSIRPαV1, hSIRPαV1(P74A), and hSIRPβ1 wasvisualized with TMB Stabilized Chromogen (Invitrogen). Reactions werestopped with 0.5 M H₂SO₄ and absorbances were read at 450 and 610 nm.EC50 values, the concentration at which 50% of the total binding signalis observed, were calculated using GraphPad Prism 6 (GraphPad Software,Inc.)

As depicted in Table 26, KWAR23, clone 18D5, and all commerciallyavailable monoclonal anti-hSIRPα antibodies are able to bind to the P74Avariant of hSIRPαV1 whereas the hSIRPα.40A and hSIRPα.50A antibodies ofthe present invention do not bind to the P74A variant of hSIRPαV1 underthe tested conditions.

TABLE 26 hSIRPαV1 hSIRPαV1(P74A) hSIRP01 binding binding bindingAntibody EC50 (nM) EC50 (nM) EC50 (nM) hSIRPα.40A 0.053 nd nd hSIRPα.50A0.307 nd nd KWAR23 0.135 0.077 0.065 18D5 0.128 0.073 0.064 anti-hSIRPα(clone SE5A5) 0.156 0.207 0.105 anti-hSIRPα (clone 7133) 0.122 0.1410.115 anti-hSIRPα (clone 106) 0.329 0.440 >2.817 anti-hSIRPα (clone 27)nd nd nd anti-hSIRPα (clone SE7C2) >7.010 >6.139 nd anti-hSIRPα (cloneP3C4) 0.179 0.197 0.160 anti-hSIRPα (clone 2A4A5) nd nd >6.456anti-hSIRPα (clone 15-414) nd nd nd anti-hSIRPα (clone 1H1) nd nd ndanti-hSIRPα (clone C-7) nd nd nd anti-hSIRPα (clone03) >8.247 >8.992 >6.092 anti-hSIRPα (clone 5E10) nd nd nd anti-hSIRPα(clone 602411) 0.047 0.076 0.051 anti-hSIRPα (cloneEPR16264) >1.166 >1.999 nd anti-hSIRPα (clone D6I3M) >6.413 >121.509 ndanti-hSIRPα (clone 001) >0.868 >1.192 nd anti-hSIRPα (cloneREA144) >3.661 >4.793 >3.075 nd, not detected

Example 27: Sequences Refererred to in the Specification

SEQ ID Description NO: SEQUENCE 50A heavy chain 1 NYYIH CDR1 (amino acidsequence) 50A heavy chain 2 WIYPGNVNTKYNEKFKA CDR2 (amino acid sequence)50A heavy chain 3 PTIIATDFDV CDR3 (amino acid sequence)50A light chain CDR1 4 KASQGVGTAVG (amino acid sequence)50A light chain CDR2 5 WASTRHT (amino acid sequence)50A light chain CDR3 6 QQYSTYPFT (amino acid sequence)humanized 50 heavy 7 EVQLX₁X₂SGX₃EX₄VKPGASVX₅X₆SCKASGFTFTNYYTHWVRQX₇Pchain variable region X₈QGLEWX₉GWIYPGNVNTKYNEKFKAX₁₀X₁₁X₁₂X₁₃TADKSTSTX₁₄(consensus sequence) YMX₁₅LSSLX₁₆SX₁₇DX₁₈AVYYCARPTITATDFDVWGQGTX₁₉VTVS Swherein: X₁ = Q, V X₂ = Q, E X₃ = A, S X₄ = V, L X₅ = K, M X₆ = V, IX₇ = A, R X₈ = G, E X₉ = I, M X₁₀ = R, K X₁₁ = V, A X₁₂ = T, IX₁₃ = I, M X₁₄ = A, V X₁₅ = D, E, Q X₁₆ = R, T X₁₇ = E, D X₁₈ = T, MX₁₉ = T, L humanized 50 light 8X₁X₂X₃X₄TQSPSX₅LSASVGDRVTITCKASQGVGTAVGWYQX₆KPGK chain variable regionX₇PKLLIYWASTRHTGVPDRFSGSGSGTX₈FTLX₉IX₁₀X₁₁LQPEDX (consensus sequence)₁₂AX₁₃YYCQQYSTYPFTFGGGTKX₁₄EIK wherein: X₁ = D, E X₂ = I, L X₃ = V, QX₄ = L, M X₅ = F, S X₆ = Q, K X₇ = A, S, V X₈ = E, D X₉ = T, AX₁₀ = S, N X₁₁ = S, N, G X₁₂ = F, I, V X₁₃ = A, D, T X₁₄ = L, VhSIRPα.50AVH1 9 GAAGTGCAGCTGCAGCAGTCTGGCGCCGAGGTCGTGAAACCTGGCG(nucleotide sequence) CCTCTGTGAAGGTGTCCTGCAAGGCCTCCGGCTTCACCTTCACCAACTACTACATCCACTGGGTGCGACAGGCCCCAGGCCAGGGACTGGAATGGATCGGCTGGATCTACCCCGGCAACGTGAACACCAAGTACAACGAGAAGTTCAAGGCCCGCGTGACCATCACCGCCGACAAGTCTACCTCCACCGCCTACATGGACCTGTCCTCCCTGAGATCCGAGGACACCGCCGTGTACTACTGCGCCAGACCCACCATCATTGCCACCGACTTCGACGTGTGGGGCCAGGGCACAACCGTGACCGTGTCCTCT hSIRPα.50AVH1 10EVQLQQSGAEVVKPGASVKVSCKASGFTFTNYYIHWVRQAPGQGLE (amino acid sequence)WIGWIYPGNVNTKYNEKFKARVTITADKSTSTAYMDLSSLRSEDTAVYYCARPTIIATDFDVWGQGTTVTVSS hSIRPα.50AVH2 11GAAGTGCAGCTGGTGGAATCCGGCTCCGAGCTCGTGAAGCCTGGCG (nucleotide sequence)CCTCCGTGAAGGTGTCCTGCAAGGCCTCTGGCTTCACCTTCACCAACTACTACATCCACTGGGTGCGACAGGCCCCAGGCCAGGGACTGGAATGGATGGGCTGGATCTACCCCGGCAACGTGAACACCAAGTACAACGAGAAGTTCAAGGCCAAGGCCACCATCACCGCCGACAAGTCCACCTCCACCGCCTACATGGAACTGTCCTCCCTGCGGAGCGAGGACACCGCCGTGTACTACTGTGCCCGGCCTACCATCATTGCCACCGACTTCGATGTGTGGGGCCAGGGCACACTCGTGACCGTGTCCTCT hSIRPα.50AVH2 12EVQLVESGSELVKPGASVKVSCKASGFTFTNYYIHWVRQAPGQGLE (amino acid sequence)WMGWIYPGNVNTKYNEKFKAKATITADKSTSTAYMELSSLRSEDTAVYYCARPTIIATDFDVWGQGTLVTVSS hSIRPα.50AVH3 13GAAGTGCAGCTGGTGCAGTCTGGCGCCGAGGTCGTGAAACCTGGCG (nucleotide sequence)CCTCCGTGATGATCTCCTGCAAGGCCTCCGGCTTCACCTTCACCAACTACTACATCCACTGGGTGCGACAGCGGCCAGGCCAGGGACTGGAATGGATCGGCTGGATCTACCCCGGCAACGTGAACACCAAGTACAACGAGAAGTTCAAGGCCCGCGTGATCATGACCGCCGACAAGTCCACCTCCACCGTGTACATGCAGCTGTCCTCCCTGACCTCCGAGGACACCGCCGTGTACTACTGCGCCAGACCCACCATCATTGCCACCGACTTCGACGTGTGGGGCCAGGGCACACTCGTGACCGTGTCCTCT hSIRPα.50AVH3 14EVQLVQSGAEVVKPGASVMISCKASGFTFTNYYIHWVRQRPGQGLE (amino acid sequence)WIGWIYPGNVNTKYNEKFKARVIMTADKSTSTVYMQLSSLTSEDTAVYYCARPTIIATDFDVWGQGTLVTVSS hSIRPα.50AVH4 15GAAGTGCAGCTGCAGCAGTCTGGCGCCGAGCTCGTGAAACCTGGCG (nucleotide sequence)CCTCTGTGAAGGTGTCCTGCAAGGCCTCCGGCTTCACCTTCACCAACTACTACATCCACTGGGTGCGACAGCGGCCAGGCCAGGGACTGGAATGGATGGGCTGGATCTACCCCGGCAACGTGAACACCAAGTACAACGAGAAGTTCAAGGCCAAGGCCACCATCACCGCCGACAAGTCCACCTCCACCGCCTACATGGAACTGTCCTCCCTGACCTCCGAGGACACCGCCGTGTACTACTGCGCCAGACCCACCATCATTGCCACCGACTTCGACGTGTGGGGCCAGGGCACAACCGTGACCGTGTCCTCT hSIRPα.50AVH4 16EVQLQQSGAELVKPGASVKVSCKASGFTFTNYYIHWVRQRPGQGLE (amino acid sequence)WMGWIYPGNVNTKYNEKFKAKATITADKSTSTAYMELSSLTSEDTAVYYCARPTIIATDFDVWGQGTTVTVSS hSIRPα.50AVH5 17GAAGTGCAGCTGGTGCAGTCTGGCGCCGAGGTCGTGAAACCTGGCG (nucleotide sequence)CCTCTGTGAAGGTGTCCTGCAAGGCCTCCGGCTTCACCTTCACCAACTACTACATCCACTGGGTGCGACAGGCCCCCGAGCAGGGACTGGAATGGATCGGCTGGATCTACCCCGGCAACGTGAACACCAAGTACAACGAGAAGTTCAAGGCCCGCGTGACCATGACCGCCGACAAGTCTACCTCCACCGCCTACATGGAACTGTCCTCCCTGCGGAGCGACGACATGGCCGTGTACTACTGCGCCAGACCCACCATCATTGCCACCGACTTCGACGTGTGGGGCCAGGGCACAACCGTGACCGTGTCCTCT hSIRPα.50AVH5 18EVQLVQSGAEVVKPGASVKVSCKASGFTFTNYYIHWVRQAPEQGLE (amino acid sequence)WIGWIYPGNVNTKYNEKFKARVTMTADKSTSTAYMELSSLRSDDMAVYYCARPTIIATDFDVWGQGTTVTVSS hSIRPα.50AVL1 19GACATCGTGCTGACCCAGTCCCCCAGCTTCCTGTCTGCCTCTGTGG (nucleotide sequence)GCGACAGAGTGACCATCACATGCAAGGCCTCTCAGGGCGTGGGCACCGCTGTGGGATGGTATCAGCAGAAGCCTGGCAAGGCCCCCAAGCTGCTGATCTACTGGGCCTCTACCAGACACACCGGCGTGCCCGACAGATTCTCCGGCTCTGGCTCTGGCACCGAGTTTACCCTGACCATCTCCAGCCTGCAGCCCGAGGATTTCGCCGCCTACTACTGCCAGCAGTACTCCACCTACCCCTTCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAG hSIRPα.50AVL1 20DIVLTQSPSFLSASVGDRVTITCKASQGVGTAVGWYQQKPGKAPKL (amino acid sequence)LIYWASTRHTGVPDRFSGSGSGTEFTLTISSLQPEDFAAYYCQQYS TYPFTFGGGTKLEIKhSIRPα.50AVL2 21 GACATCGTGATGACCCAGTCCCCCTCCAGCCTGTCTGCCTCTGTGG(nucleotide sequence) GCGACAGAGTGACCATCACATGCAAGGCCTCTCAGGGCGTGGGCACCGCTGTGGGATGGTATCAGCAGAAGCCTGGCAAGGCCCCCAAGCTGCTGATCTACTGGGCCTCTACCAGACACACCGGCGTGCCCGACAGATTCTCCGGCTCTGGCTCTGGCACCGACTTCACCCTGACCATCTCCAACCTGCAGCCCGAGGACTTCGCCGACTACTACTGCCAGCAGTACTCCACCTACCCCTTCACCTTCGGCGGAGGCACCAAGGTGGAAATCAAG hSIRPα.50AVL2 22DIVMTQSPSSLSASVGDRVTITCKASQGVGTAVGWYQQKPGKAPKL (amino acid sequence)LIYWASTRHTGVPDRFSGSGSGTDFTLTISNLQPEDFADYYCQQYS TYPFTFGGGTKVEIKhSIRPα.50AVL3 23 GAGCTCGTGATGACCCAGTCCCCTTCCAGCCTGTCTGCCTCCGTGG(nucleotide sequence) GCGACAGAGTGACCATCACATGCAAGGCCTCTCAGGGCGTGGGCACCGCTGTGGGATGGTATCAGCAGAAGCCTGGCAAGGCCCCCAAGCTGCTGATCTACTGGGCCTCTACCAGACACACCGGCGTGCCCGACAGATTCTCCGGCTCTGGCTCTGGCACCGACTTTACCCTGGCCATCTCCAGCCTGCAGCCCGAGGATATCGCCGACTACTACTGCCAGCAGTACTCCACCTACCCCTTCACCTTCGGCGGAGGCACCAAGGTGGAAATCAAG hSIRPα.50AVL3 24ELVMTQSPSSLSASVGDRVTITCKASQGVGTAVGWYQQKPGKAPKL (amino acid sequence)LIYWASTRHTGVPDRFSGSGSGTDFTLAISSLQPEDIADYYCQQYS TYPFTFGGGTKVEIKhSIRPα.50AVL4 25 GACATCCAGATGACCCAGTCCCCCTCCAGCCTGTCTGCCTCTGTGG(nucleotide sequence) GCGACAGAGTGACCATCACATGCAAGGCCTCTCAGGGCGTGGGCACCGCTGTGGGCTGGTATCAGAAAAAGCCCGGCAAGGTGCCCAAGCTGCTGATCTACTGGGCCTCCACCAGACACACCGGCGTGCCCGATAGATTCTCCGGCTCTGGCTCTGGCACCGACTTCACCCTGACCATCAACGGCCTGCAGCCTGAGGACGTGGCCACCTACTACTGCCAGCAGTACTCCACCTACCCCTTCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAG hSIRPα.50AVL4 26DIQMTQSPSSLSASVGDRVTITCKASQGVGTAVGWYQKKPGKVPKL (amino acid sequence)LIYWASTRHTGVPDRFSGSGSGTDFTLTINGLQPEDVATYYCQQYS TYPFTFGGGTKLEIKhSIRPα.50AVL5 27 GACATCGTGCTGACCCAGTCCCCCAGCTTCCTGTCTGCCTCTGTGG(nucleotide sequence) GCGACAGAGTGACCATCACATGCAAGGCCTCTCAGGGCGTGGGCACCGCTGTGGGATGGTATCAGCAGAAGCCCGGCAAGTCCCCCAAGCTGCTGATCTACTGGGCCTCCACCAGACACACCGGCGTGCCCGATAGATTCTCCGGCTCTGGCTCTGGCACCGAGTTCACCCTGACCATCTCCAACCTGCAGCCCGAGGACTTCGCCGCCTACTACTGCCAGCAGTACTCCACCTACCCCTTCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAG hSIRPα.50AVL5 28DIVLTQSPSFLSASVGDRVTITCKASQGVGTAVGWYQQKPGKSPKL (amino acid sequence)LIYWASTRHTGVPDRFSGSGSGTEFTLTISNLQPEDFAAYYCQQYS TYPFTFGGGTKLEIKhSIRPα.50A mouse 29 CAGGTCCAGCTGCAGCAGTCTGGACCTGAACTGGTGAAGCCTGGGGVH (nucleotide CTTCAGTTAGGATATCCTGCAAGGCTTCTGGCTTCACCTTCACAAA sequence)CTACTATATACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAAATGTTAATACTAAGTACAATGAGAAGTTCAAGGCCAAGGCCACACTGACTGCAGACAAATCCTCCACCACAGCCTACATGCAGCTCAGCAGCCTGGCCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGACCTACGATAATAGCTACGGACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA hSIRPα.50A mouse 30QVQLQQSGPELVKPGASVRISCKASGFTFTNYYIHWVKQRPGQGLE VH (amino acidWIGWIYPGNVNTKYNEKFKAKATLTADKSSTTAYMQLSSLASEDSA sequence)VYFCARPTIIATDFDVWGAGTTVTVSS hSIRPα.50A mouse 31GACATTGTCATGACCCAGTCTCACAAATTCATGTCCACATCAGTAG VL (nucleotideGAGACAGGGTCAACATCACCTGCAAGGCCAGTCAGGGTGTGGGTAC sequence)TGCTGTAGGCTGGTATCAACAGAAACCAGGGCAATCTCCTAGACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCAGTCTCGCCATTAGCAATGTGCAGTCTGAAGACCTGGCAGATTATTTCTGTCAGCAATATAGCACCTATCCGTTCACGTTCGGAGGGGGGACCAATCTAGAAATAAAA hSIRPα.50A mouse 32DIVMTQSHKFMSTSVGDRVNITCKASQGVGTAVGWYQQKPGQSPRL VL (amino acidLIYWASTRHTGVPDRFTGSGSGTDFSLAISNVQSEDLADYFCQQYS sequence) TYPFTFGGGTNLEIKhuman SIRPαV1 33 ATGGAGCCCGCCGGCCCGGCCCCCGGCCGCCTCGGGCCGCTGCTCT(nucleotide sequence) GCCTGCTGCTCGCCGCGTCCTGCGCCTGGTCAGGAGTGGCGGGTGAGGAGGAGCTGCAGGTGATTCAGCCTGACAAGTCCGTGTTGGTTGCAGCTGGAGAGACAGCCACTCTGCGCTGCACTGCGACCTCTCTGATCCCTGTGGGGCCCATCCAGTGGTTCAGAGGAGCTGGACCAGGCCGGGAATTAATCTACAATCAAAAAGAAGGCCACTTCCCCCGGGTAACAACTGTTTCAGACCTCACAAAGAGAAACAACATGGACTTTTCCATCCGCATCGGTAACATCACCCCAGCAGATGCCGGCACCTACTACTGTGTGAAGTTCCGGAAAGGGAGCCCCGATGACGTGGAGTTTAAGTCTGGAGCAGGCACTGAGCTGTCTGTGCGCGCCAAACCCTCTGCCCCCGTGGTATCGGGCCCTGCGGCGAGGGCCACACCTCAGCACACAGTGAGCTTCACCTGCGAGTCCCACGGCTTCTCACCCAGAGACATCACCCTGAAATGGTTCAAAAATGGGAATGAGCTCTCAGACTTCCAGACCAACGTGGACCCCGTAGGAGAGAGCGTGTCCTACAGCATCCACAGCACAGCCAAGGTGGTGCTGACCCGCGAGGACGTTCACTCTCAAGTCATCTGCGAGGTGGCCCACGTCACCTTGCAGGGGGACCCTCTTCGTGGGACTGCCAACTTGTCTGAGACCATCCGAGTTCCACCCACCTTGGAGGTTACTCAACAGCCCGTGAGGGCAGAGAACCAGGTGAATGTCACCTGCCAGGTGAGGAAGTTCTACCCCCAGAGACTACAGCTGACCTGGTTGGAGAATGGAAACGTGTCCCGGACAGAAACGGCCTCAACCGTTACAGAGAACAAGGATGGTACCTACAACTGGATGAGCTGGCTCCTGGTGAATGTATCTGCCCACAGGGATGATGTGAAGCTCACCTGCCAGGTGGAGCATGACGGGCAGCCAGCGGTCAGCAAAAGCCATGACCTGAAGGTCTCAGCCCACCCGAAGGAGCAGGGCTCAAATACCGCCGCTGAGAACACTGGATCTAATGAACGGAACATCTATATTGTGGTGGGTGTGGTGTGCACCTTGCTGGTGGCCCTACTGATGGCGGCCCTCTACCTCGTCCGAATCAGACAGAAGAAAGCCCAGGGCTCCACTTCTTCTACAAGGTTGCATGAGCCCGAGAAGAATGCCAGAGAAATAACACAGGACACAAATGATATCACATATGCAGACCTGAACCTGCCCAAGGGGAAGAAGCCTGCTCCCCAGGCTGCGGAGCCCAACAACCACACGGAGTATGCCAGCATTCAGACCAGCCCGCAGCCCGCGTCGGAGGACACCCTCACCTATGCTGACCTGGACATGGTCCACCTCAACCGGACCCCCAAGCAGCCGGCCCCCAAGCCTGAGCCGTCCTTCTCAGAGTACGCCAGCGTCCAGGTCCCGAGGAAG human SIRPαV1 34MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVA (amino acid sequence)AGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human SIRPαV2 35ATGGAACCTGCCGGACCTGCCCCTGGCAGACTGGGACCTCTGCTGT (nucleotide sequence)GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGAAGAGGAACTGCAAGTGATCCAGCCCGACAAGAGCGTGTCCGTGGCTGCTGGCGAGTCTGCCATCCTGCACTGTACCGTGACCAGCCTGATCCCCGTGGGCCCCATCCAGTGGTTTAGAGGCGCTGGCCCTGCCAGAGAGCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACCGTGTCCGAGAGCACCAAGCGCGAGAACATGGACTTCAGCATCAGCATCTCCAACATCACCCCTGCCGACGCCGGCACCTACTACTGCGTGAAGTTCAGAAAGGGCAGCCCCGACACCGAGTTCAAGAGCGGCGCTGGAACCGAGCTGTCTGTGCGGGCTAAGCCTTCTGCCCCTGTGGTGTCTGGACCTGCCGCCAGAGCTACACCTCAGCACACCGTGTCTTTCACATGCGAGAGCCACGGCTTCAGCCCCAGAGACATCACCCTGAAGTGGTTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACCCTGTGGGCGAGTCCGTGTCCTACAGCATCCACAGCACCGCCAAGGTGGTGCTGACCCGCGAGGATGTGCACAGCCAAGTGATCTGCGAGGTGGCCCACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCTAACCTGAGCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCCCGTGCGGGCTGAGAACCAAGTGAACGTGACCTGCCAAGTGCGGAAGTTCTACCCTCAGAGACTGCAGCTGACCTGGCTGGAAAACGGAAACGTGTCCAGAACCGAGACAGCCAGCACCGTGACAGAGAACAAGGACGGCACATACAACTGGATGAGCTGGCTGCTCGTGAACGTGTCCGCCCACAGAGATGACGTGAAGCTGACATGCCAGGTGGAACACGACGGCCAGCCTGCCGTGTCTAAGAGCCACGACCTGAAGGTGTCCGCTCACCCCAAAGAGCAGGGCAGCAACACCGCCGCTGAGAACACAGGCAGCAACGAGAGAAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGGTGGCTCTGCTGATGGCTGCCCTGTACCTCGTGCGGATCAGACAGAAGAAGGCCCAGGGCTCCACCTCCAGCACCAGACTGCACGAGCCTGAGAAGAACGCCCGCGAGATCACCCAGGACACCAACGACATCACCTACGCCGACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCTGCCGAGCCTAACAACCACACAGAGTACGCCAGCATCCAGACCAGCCCTCAGCCTGCCAGCGAGGACACACTGACATACGCCGATCTGGACATGGTGCACCTGAACAGAACCCCCAAGCAGCCCGCTCCCAAGCCCGAGCCTAGCTTCTCTGAGTACGCCTCCGTGCAGGTGCCCAGAAAA human SIRPαV2 36MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVSVA (amino acid sequence)AGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human SIRPβ1 37ATGCCCGTGCCAGCCTCCTGGCCCCACCTTCCTAGTCCTTTCCTGC (nucleotide sequence)TGATGACGCTACTGCTGGGGAGACTCACAGGAGTGGCAGGTGAGGACGAGCTACAGGTGATTCAGCCTGAAAAGTCCGTATCAGTTGCAGCTGGAGAGTCGGCCACTCTGCGCTGTGCTATGACGTCCCTGATCCCTGTGGGGCCCATCATGTGGTTTAGAGGAGCTGGAGCAGGCCGGGAATTAATCTACAATCAGAAAGAAGGCCACTTCCCACGGGTAACAACTGTTTCAGAACTCACAAAGAGAAACAACCTGGACTTTTCCATCAGCATCAGTAACATCACCCCAGCAGACGCCGGCACCTACTACTGTGTGAAGTTCCGGAAAGGGAGCCCTGACGACGTGGAGTTTAAGTCTGGAGCAGGCACTGAGCTGTCTGTGCGCGCCAAACCCTCTGCCCCCGTGGTATCGGGCCCTGCGGTGAGGGCCACACCTGAGCACACAGTGAGCTTCACCTGCGAGTCCCATGGCTTCTCTCCCAGAGACATCACCCTGAAATGGTTCAAAAATGGGAATGAGCTCTCAGACTTCCAGACCAACGTGGACCCCGCAGGAGACAGTGTGTCCTACAGCATCCACAGCACAGCCAGGGTGGTGCTGACCCGTGGGGACGTTCACTCTCAAGTCATCTGCGAGATAGCCCACATCACCTTGCAGGGGGACCCTCTTCGTGGGACTGCCAACTTGTCTGAGGCCATCCGAGTTCCACCCACCTTGGAGGTTACTCAACAGCCCATGAGGGCAGAGAACCAGGCAAACGTCACCTGCCAGGTGAGCAATTTCTACCCCCGGGGACTACAGCTGACCTGGTTGGAGAATGGAAATGTGTCCCGGACAGAAACAGCTTCGACCCTCATAGAGAACAAGGATGGCACCTACAACTGGATGAGCTGGCTCCTGGTGAACACCTGTGCCCACAGGGACGATGTGGTGCTCACCTGTCAGGTGGAGCATGATGGGCAGCAAGCAGTCAGCAAAAGCTATGCCCTGGAGATCTCAGCGCACCAGAAGGAGCACGGCTCAGATATCACCCATGAAGCAGCGCTGGCTCCTACTGCTCCACTCCTCGTAGCTCTCCTCCTGGGCCCCAAGCTGCTACTGGTGGTTGGTGTCTCTGCCATCTACATCTGCTGGAAACAGAAGGCC human SIRPβ1 (amino 38MPVPASWPHLPSPFLLMTLLLGRLTGVAGEDELQVIQPEKSVSVAA acid sequence)GESATLRCAMTSLIPVGPIMWFRGAGAGRELIYNQKEGHFPRVTTVSELTKRNNLDFSISISNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAVRATPEHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPAGDSVSYSIHSTARVVLTRGDVHSQVICEIAHITLQGDPLRGTANLSEAIRVPPTLEVTQQPMRAENQANVTCQVSNFYPRGLQLTWLENGNVSRTETASTLIENKDGTYNWMSWLLVNTCAHRDDVVLTCQVEHDGQQAVSKSYALEISAHQKEHGSDITHEAALAPTAPLLVALLLGPKLLLVVGVSAIYICWKQKA human SIRPγ 39ATGCCTGTCCCAGCCTCCTGGCCCCATCCTCCTGGTCCTTTCCTGC (nucleotide sequence)TTCTGACTCTACTGCTGGGACTTACAGAAGTGGCAGGTGAGGAGGAGCTACAGATGATTCAGCCTGAGAAGCTCCTGTTGGTCACAGTTGGAAAGACAGCCACTCTGCACTGCACTGTGACCTCCCTGCTTCCCGTGGGACCCGTCCTGTGGTTCAGAGGAGTTGGACCAGGCCGGGAATTAATCTACAATCAAAAAGAAGGCCACTTCCCCAGGGTAACAACAGTTTCAGACCTCACAAAGAGAAACAACATGGACTTTTCCATCCGCATCAGTAGCATCACCCCAGCAGATGTCGGCACATACTACTGTGTGAAGTTTCGAAAAGGGAGCCCTGAGAACGTGGAGTTTAAGTCTGGACCAGGCACTGAGATGGCTTTGGGTGCCAAACCCTCTGCCCCCGTGGTATTGGGCCCTGCGGCGAGGACCACACCTGAGCATACAGTGAGTTTCACCTGTGAGTCCCATGGCTTCTCTCCCAGAGACATCACCCTGAAATGGTTCAAAAATGGGAATGAGCTCTCAGACTTCCAGACCAACGTGGACCCCACAGGACAGAGTGTGGCCTACAGCATCCGCAGCACAGCCAGGGTGGTACTGGACCCCTGGGACGTTCGCTCTCAGGTCATCTGCGAGGTGGCCCATGTCACCTTGCAGGGGGACCCTCTTCGTGGGACTGCCAACTTGTCTGAGGCCATCCGAGTTCCACCCACCTTGGAGGTTACTCAACAGCCCATGAGGGTGGGGAACCAGGTAAACGTCACCTGCCAGGTGAGGAAGTTCTACCCCCAGAGCCTACAGCTGACCTGGTCGGAGAATGGAAACGTGTGCCAGAGAGAAACAGCCTCGACCCTTACAGAGAACAAGGATGGTACCTACAACTGGACAAGCTGGTTCCTGGTGAACATATCTGACCAAAGGGATGATGTGGTCCTCACCTGCCAGGTGAAGCATGATGGGCAGCTGGCGGTCAGCAAACGCCTTGCCCTAGAGGTCACAGTCCACCAGAAGGACCAGAGCTCAGATGCTACCCCTGGCCCGGCATCATCCCTTACTGCGCTGCTCCTCATAGCTGTCCTCCTGGGCCCCATCTACGTCCCCTGGA AGCAGAAGACChuman SIRPγ (amino 40 MPVPASWPHPPGPFLLLTLLLGLTEVAGEEELQMIQPEKLLLVTVGacid sequence) KTATLHCTVTSLLPVGPVLWFRGVGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPENVEFKSGPGTEMALGAKPSAPVVLGPAARTTPEHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPTGQSVAYSIRSTARVVLDPWDVRSQVICEVAHVTLQGDPLRGTANLSEAIRVPPTLEVTQQPMRVGNQVNVTCQVRKFYPQSLQLTWSENGNVCQRETASTLTENKDGTYNWTSWFLVNISDQRDDVVLTCQVKHDGQLAVSKRLALEVTVHQKDQSSDATPGPASSLTA LLLIAVLLGPIYVPWKQKThuman CD47 41 ATGTGGCCTCTGGTGGCCGCTCTGCTGCTGGGCTCTGCTTGTTGTG(nucleotide sequence) GATCCGCCCAGCTGCTGTTCAACAAGACCAAGTCCGTGGAGTTCACCTTCTGCAACGATACCGTCGTGATCCCCTGCTTCGTGACCAACATGGAAGCCCAGAACACCACCGAGGTGTACGTGAAGTGGAAGTTCAAGGGCCGGGACATCTACACCTTCGACGGCGCCCTGAACAAGTCCACCGTGCCCACCGATTTCTCCAGCGCCAAGATCGAGGTGTCACAGCTGCTGAAGGGCGACGCCTCCCTGAAGATGGACAAGTCCGACGCCGTGTCCCACACCGGCAACTACACCTGTGAAGTGACCGAGCTGACCAGAGAGGGCGAGACAATCATCGAGCTGAAGTACCGGGTGGTGTCCTGGTTCAGCCCCAACGAGAACATCCTGATCGTGATCTTCCCCATCTTCGCCATCCTGCTGTTCTGGGGCCAGTTCGGCATCAAGACCCTGAAGTACAGATCCGGCGGCATGGACGAAAAGACAATCGCCCTGCTGGTGGCTGGCCTCGTGATCACCGTGATTGTGATCGTGGGCGCTATCCTGTTCGTGCCCGGCGAGTACAGCCTGAAGAATGCTACCGGCCTGGGCCTGATTGTGACCTCCACCGGAATCCTGATCCTGCTGCACTACTACGTGTTCTCCACCGCTATCGGCCTGACCTCCTTCGTGATCGCCATTCTCGTGATCCAAGTGATCGCCTACATCCTGGCCGTCGTGGGCCTGTCCCTGTGTATCGCCGCCTGCATCCCTATGCACGGCCCCCTGCTGATCTCCGGCCTGTCTATTCTGGCCCTGGCTCAGCTGCTGGGACTGGTGTACATGAAGTTCGTGGCCTCCAACCAGAAAACCATCCAGCCCCCTCGGAAGGCCGTGGAAGAACCCCTGAACGCCTTCAAAGAATCCAAGGGCATGATGAACGAC GAA human CD47 (amino 42MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNM acid sequence)EAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMND E human SIRPαV3 43ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT (nucleotide sequence)GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGAAGAGGAACTGCAAGTGATCCAGCCCGACAAGTCCGTGTCTGTGGCCGCTGGCGAGTCTGCCATCCTGCTGTGTACCGTGACCTCCCTGATCCCCGTGGGCCCCATCCAGTGGTTTAGAGGCGCTGGCCCTGCCAGAGAGCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACCGTGTCCGAGTCCACCAAGCGCGAGAACATGGACTTCTCCATCTCCATCAGCAACATCACCCCTGCCGACGCCGGCACCTACTACTGCGTGAAGTTCCGGAAGGGCTCCCCCGACACCGAGTTCAAGTCTGGCGCTGGCACCGAGCTGTCTGTGCGGGCTAAACCTTCTGCCCCTGTGGTGTCTGGACCTGCCGCTAGAGCTACCCCTCAGCACACCGTGTCTTTTACCTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGGTTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACCCTGTGGGCGAGAGCGTGTCCTACTCCATCCACTCCACCGCCAAGGTGGTGCTGACACGCGAGGACGTGCACTCCCAAGTGATCTGCGAGGTGGCCCACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAACCTGTCCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCCAGTGCGGGCCGAGAACCAAGTGAACGTGACCTGCCAAGTGCGGAAGTTCTACCCCCAGCGGCTGCAGCTGACCTGGCTGGAAAACGGCAATGTGTCCCGGACCGAGACAGCCAGCACCGTGACCGAGAACAAGGATGGCACCTACAATTGGATGTCTTGGCTGCTCGTGAACGTGTCCGCCCACCGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGACGGCCAGCCTGCCGTGTCCAAGAGCCACGATCTGAAGGTGTCCGCTCATCCCAAAGAGCAGGGCTCCAACACCGCCGCTGAGAACACCGGCTCTAACGAGCGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGGTGGCTCTGCTGATGGCTGCCCTGTACCTCGTGCGGATCCGGCAGAAGAAGGCCCAGGGCTCTACCTCCTCCACCAGACTGCACGAGCCCGAGAAGAACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACGCCGACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCTGCCGAGCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCTCAGCCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGGTGCACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCCTAGCTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA human SIRPαV3 44MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVSVA (amino acid sequence)AGESAILLCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human SIRPαV4 45ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT (nucleotide sequence)GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGAAGAGGGCCTGCAAGTGATCCAGCCCGACAAGTCCGTGTCTGTGGCCGCTGGCGAGTCTGCCATCCTGCACTGTACCGCCACCTCCCTGATCCCCGTGGGACCCATCCAGTGGTTTAGAGGCGCTGGCCCTGGCAGAGAGCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACCGTGTCCGACCTGACCAAGCGGAACAACATGGACTTCTCCATCCGGATCGGCAACATCACCCCTGCCGATGCCGGCACCTACTACTGCGTGAAGTTCCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCTGGCACCGAGCTGTCTGTGCGGGCTAAACCTTCTGCCCCTGTGGTGTCTGGCCCTGCCGCTAGAGCTACCCCTCAGCACACCGTGTCTTTTACCTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGGTTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACCCTGTGGGCGAGAGCGTGTCCTACTCCATCCACTCCACCGCCAAGGTGGTGCTGACACGCGAGGACGTGCACTCCCAAGTGATCTGCGAGGTGGCCCACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAACCTGTCCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCCAGTGCGGGCCGAGAACCAAGTGAACGTGACCTGCCAAGTGCGGAAGTTCTACCCCCAGCGGCTGCAGCTGACCTGGCTGGAAAACGGCAATGTGTCCCGGACCGAGACAGCCTCCACCGTGACCGAGAACAAGGATGGCACCTACAATTGGATGTCTTGGCTGCTCGTGAACGTGTCCGCCCACCGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGACGGCCAGCCTGCCGTGTCCAAGTCCCACGATCTGAAGGTGTCCGCTCATCCCAAAGAGCAGGGCTCCAACACCGCCGCTGAGAACACCGGCTCTAACGAGCGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGGTGGCTCTGCTGATGGCTGCCCTGTACCTCGTGCGGATCCGGCAGAAGAAGGCCCAGGGCTCTACCTCCTCCACCAGACTGCACGAGCCCGAGAAGAACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACGCCGACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCTGCCGAGCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCTCAGCCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGGTGCACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCCTAGCTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA human SIRPαV4 46MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEGLQVIQPDKSVSVA (amino acid sequence)AGESAILHCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human SIRPαV5 47ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT (nucleotide sequence)GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGAAGAGGAACTGCAAGTGATCCAGCCCGACAAGTTCGTGCTGGTGGCCGCTGGCGAGACAGCCACCCTGAGATGTACCGCCACCTCCCTGATCCCCGTGGGCCCTATCCAGTGGTTTAGAGGCGCTGGCCCTGGCAGAGAGCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACCGTGTCCGACCTGACCAAGCGGAACAACATGGACTTCTCCATCCGGATCGGCAACATCACCCCTGCCGATGCCGGCACCTACTACTGCGTGAAGTTCCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCTGGCACCGAGCTGTCTGTGCGGGCTAAACCTTCTGCCCCTGTGGTGTCTGGCCCTGCCGCTAGAGCTACCCCTCAGCACACCGTGTCTTTTACCTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGGTTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACCCTGTGGGCGAGTCCGTGTCCTACTCCATCCACTCCACCGCCAAGGTGGTGCTGACACGCGAGGACGTGCACTCCCAAGTGATCTGCGAGGTGGCCCACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAACCTGTCCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCCAGTGCGGGCCGAGAACCAAGTGAACGTGACCTGCCAAGTGCGGAAGTTCTACCCCCAGCGGCTGCAGCTGACCTGGCTGGAAAACGGCAATGTGTCCCGGACCGAGACTGCCTCCACCGTGACCGAGAACAAGGATGGCACCTACAATTGGATGTCTTGGCTGCTCGTGAACGTGTCCGCCCACCGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGACGGCCAGCCTGCCGTGTCCAAGTCCCACGATCTGAAGGTGTCCGCTCATCCCAAAGAGCAGGGCTCCAACACCGCCGCTGAGAACACCGGCTCTAACGAGCGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGGTGGCTCTGCTGATGGCTGCCCTGTACCTCGTGCGGATCCGGCAGAAGAAGGCCCAGGGCTCTACCTCCTCCACCAGACTGCACGAGCCCGAGAAGAACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACGCCGACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCTGCCGAGCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCTCAGCCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGGTGCACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCCTAGCTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA human SIRPαV5 48MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKFVLVA (amino acid sequence)AGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human SIRPαV6 49ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT (nucleotide sequence)GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGAAGAGGAACTGCAAGTGATCCAGCCCGACAAGTCCGTGCTGGTGGCTGCTGGCGAGACTGCCACCCTGAGATGTACCGCCACCTCCCTGATCCCCGTGGGCCCTATCCAGTGGTTTAGAGGCGCTGGCCCTGGCAGAGAGCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACCGTGTCCGACCTGACCAAGCGGAACAACATGGACTTCCCCATCCGGATCGGCAACATCACCCCTGCCGATGCCGGCACCTACTACTGCGTGAAGTTCCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCTGGCACCGAGCTGTCTGTGCGGGCTAAACCTTCTGCCCCTGTGGTGTCTGGCCCTGCCGCTAGAGCTACCCCTCAGCACACCGTGTCTTTTACCTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGGTTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACCCTGTGGGCGAGTCCGTGTCCTACTCCATCCACTCCACCGCCAAGGTGGTGCTGACACGCGAGGACGTGCACTCCCAAGTGATCTGCGAGGTGGCCCACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAACCTGTCCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCCCGTGCGGGCTGAGAACCAAGTGAACGTGACCTGCCAAGTGCGGAAGTTCTACCCCCAGCGGCTGCAGCTGACCTGGCTGGAAAACGGCAATGTGTCCCGGACCGAGACAGCCTCCACCGTGACCGAGAACAAGGATGGCACCTACAATTGGATGTCCTGGCTGCTCGTGAACGTGTCCGCCCACCGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGACGGCCAGCCTGCCGTGTCCAAGTCCCACGATCTGAAGGTGTCCGCTCATCCCAAAGAGCAGGGCTCCAACACCGCCGCTGAGAACACCGGCTCTAACGAGCGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGGTGGCACTGCTGATGGCCGCTCTGTACCTCGTGCGGATCCGGCAGAAGAAGGCCCAGGGCTCTACCTCCTCCACCAGACTGCACGAGCCCGAGAAGAACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACGCCGACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCTGCCGAGCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCTCAGCCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGGTGCACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCCTAGCTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA human SIRPαV6 50MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVA (amino acid sequence)AGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFPIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human SIRPαV8 51ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT (nucleotide sequence)GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGAAGAGGAACTGCAAGTGATCCAGCCCGACAAGTCCGTGCTGGTGGCTGCTGGCGAGACTGCCACCCTGAGATGTACCGCCACCTCCCTGATCCCCGTGGGCCCTATCCAGTGGTTTAGAGGCGCTGGCCCTGCCAGAGAGCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACCGTGTCCGAGTCCACCAAGCGCGAGAACATGGACTTCTCCATCTCCATCAGCAACATCACCCCTGCCGACGCCGGCACCTACTACTGCGTGAAGTTCCGGAAGGGCTCCCCCGACACCGAGTTCAAGTCTGGCGCTGGCACCGAGCTGTCTGTGCGGGCTAAACCTTCTGCCCCTGTGGTGTCTGGACCTGCCGCTAGAGCTACCCCTCAGCACACCGTGTCTTTTACCTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGGTTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACCCTGTGGGCGAGTCCGTGTCCTACTCCATCCACTCCACCGCCAAGGTGGTGCTGACACGCGAGGACGTGCACTCCCAAGTGATCTGCGAGGTGGCCCACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAACCTGTCCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCCCGTGCGGGCTGAGAACCAAGTGAACGTGACCTGCCAAGTGCGGAAGTTCTACCCCCAGCGGCTGCAGCTGACCTGGCTGGAAAACGGCAATGTGTCCCGGACCGAGACAGCCAGCACCGTGACCGAGAACAAGGATGGCACCTACAATTGGATGTCCTGGCTGCTCGTGAACGTGTCCGCCCACCGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGACGGCCAGCCTGCCGTGTCCAAGAGCCACGATCTGAAGGTGTCCGCTCATCCCAAAGAGCAGGGCTCCAACACCGCCGCTGAGAACACCGGCTCTAACGAGCGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGGTGGCACTGCTGATGGCCGCTCTGTACCTCGTGCGGATCCGGCAGAAGAAGGCCCAGGGCTCTACCTCCTCCACCAGACTGCACGAGCCCGAGAAGAACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACGCCGACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCTGCCGAGCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCTCAGCCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGGTGCACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCCTAGCTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA human SIRPαV8 52MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVA (amino acid sequence)AGETATLRCTATSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human SIRPαV9 53ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT (nucleotide sequence)GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGAAGAGGAACTGCAAGTGATCCAGCCCGACAAGTCCGTGCTGGTGGCTGCTGGCGAGACTGCCACCCTGAGATGTACCGCCACCTCCCTGATCCCCGTGGGCCCTATCCAGTGGTTTAGAGGCGCTGGCCCTGGCAGAGAGCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACCGTGTCCGACCTGACCAAGCGGAACAACATGGACTTCTCCATCCGGATCTCCAACATCACCCCTGCCGACGCCGGCACCTACTACTGCGTGAAGTTCCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCTGGCACCGAGCTGTCTGTGCGGGCTAAACCTTCTGCCCCTGTGGTGTCTGGCCCTGCCGCTAGAGCTACCCCTCAGCACACCGTGTCTTTTACCTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGGTTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACCCTGTGGGCGAGTCCGTGTCCTACTCCATCCACTCCACCGCCAAGGTGGTGCTGACACGCGAGGACGTGCACTCCCAAGTGATCTGCGAGGTGGCCCACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAACCTGTCCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCCCGTGCGGGCTGAGAACCAAGTGAACGTGACCTGCCAAGTGCGGAAGTTCTACCCCCAGCGGCTGCAGCTGACCTGGCTGGAAAACGGCAATGTGTCCCGGACCGAGACAGCCTCCACCGTGACCGAGAACAAGGATGGCACCTACAATTGGATGTCCTGGCTGCTCGTGAACGTGTCCGCCCACCGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGACGGCCAGCCTGCCGTGTCCAAGTCCCACGATCTGAAGGTGTCCGCTCATCCCAAAGAGCAGGGCTCCAACACCGCCGCTGAGAACACCGGCTCTAACGAGCGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGGTGGCACTGCTGATGGCCGCTCTGTACCTCGTGCGGATCCGGCAGAAGAAGGCCCAGGGCTCTACCTCCTCCACCAGACTGCACGAGCCCGAGAAGAACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACGCCGACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCTGCCGAGCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCTCAGCCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGGTGCACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCCTAGCTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA human SIRPαV9 54MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVA (amino acid sequence)AGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK hSIRPα-VβC1αC2α 55ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT (nucleotide sequence)GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGAGGACGAGCTGCAAGTGATCCAGCCCGAGAAGTCCGTGTCTGTGGCCGCTGGCGAGTCTGCCACCCTGAGATGCGCTATGACCTCCCTGATCCCCGTGGGCCCCATCATGTGGTTTAGAGGCGCTGGCGCTGGCAGAGAGCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACCGTGTCCGAGCTGACCAAGCGGAACAACCTGGACTTCTCCATCTCCATCAGCAACATCACCCCTGCCGACGCCGGCACCTACTACTGCGTGAAGTTCCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCTGGAACCGAGCTGTCCGTGCGGGCTAAACCTTCTGCCCCTGTGGTGTCTGGCCCTGCCGCTAGAGCTACCCCTCAGCACACCGTGTCTTTTACCTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGGTTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACCCTGTGGGCGAGAGCGTGTCCTACTCCATCCACTCCACCGCCAAGGTGGTGCTGACACGCGAGGACGTGCACTCCCAAGTGATCTGCGAGGTGGCCCACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAACCTGTCCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCCTGTGCGGGCCGAGAACCAAGTGAACGTGACCTGCCAAGTGCGGAAGTTCTACCCCCAGCGGCTGCAGCTGACCTGGCTGGAAAACGGCAATGTGTCCCGGACCGAGACAGCCAGCACCGTGACCGAGAACAAGGATGGCACCTACAATTGGATGTCCTGGCTGCTCGTGAACGTGTCCGCCCACCGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGACGGCCAGCCTGCCGTGTCCAAGTCCCACGATCTGAAGGTGTCCGCTCATCCCAAAGAGCAGGGCTCCAACACCGCCGCTGAGAACACCGGCTCTAACGAGCGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGGTGGCTCTGCTGATGGCTGCCCTGTACCTCGTGCGGATCCGGCAGAAGAAGGCCCAGGGCTCTACCTCCTCCACCAGACTGCACGAGCCTGAGAAGAACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACGCCGACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCCGCCGAGCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCTCAGCCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGGTGCACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCCTAGCTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA hSIRPα-VβC1αC2α 56MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEDELQVIQPEKSVSVA (amino acid sequence)AGESATLRCAMTSLIPVGPIMWFRGAGAGRELIYNQKEGHFPRVTTVSELTKRNNLDFSISISNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK hSIRPα-VαC1βC2α 57ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT (nucleotide sequence)GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGAAGAGGAACTGCAAGTGATCCAGCCCGACAAGTCCGTGCTGGTGGCTGCTGGCGAGACTGCCACCCTGAGATGTACCGCCACCTCCCTGATCCCCGTGGGCCCTATCCAGTGGTTTAGAGGCGCTGGCCCTGGCAGAGAGCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACCGTGTCCGACCTGACCAAGCGGAACAACATGGACTTCTCCATCCGGATCGGCAACATCACCCCTGCCGATGCCGGCACCTACTACTGCGTGAAGTTCCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCTGGCACCGAGCTGTCTGTGCGGGCTAAACCTTCTGCCCCCGTGGTGTCTGGACCTGCCGTGCGAGCTACCCCTGAGCACACCGTGTCTTTTACCTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGGTTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACCCAGCCGGCGACTCCGTGTCCTACTCCATCCACTCTACCGCCAGAGTGGTGCTGACCAGAGGCGACGTGCACTCCCAAGTGATCTGCGAGATCGCCCATATCACACTGCAGGGCGACCCCCTGAGAGGCACCGCTAACCTGTCTGAGACAATCCGGGTGCCCCCCACCCTGGAAGTGACTCAGCAGCCAGTGCGGGCCGAGAACCAAGTGAACGTGACCTGCCAAGTGCGGAAGTTCTACCCCCAGCGGCTGCAGCTGACCTGGCTGGAAAACGGCAATGTGTCCCGGACCGAGACAGCCTCCACCGTGACCGAGAACAAGGATGGCACCTACAATTGGATGTCTTGGCTGCTCGTGAACGTGTCCGCCCACCGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGACGGCCAGCCTGCCGTGTCCAAGTCCCACGATCTGAAGGTGTCCGCTCATCCCAAAGAGCAGGGCTCCAACACCGCCGCTGAGAACACCGGCTCTAACGAGCGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGGTGGCACTGCTGATGGCCGCTCTGTACCTCGTGCGGATCCGGCAGAAGAAGGCCCAGGGCTCTACCTCCTCCACCAGACTGCACGAGCCCGAGAAGAACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACGCCGACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCCGCCGAGCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCTCAGCCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGGTGCACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCCTAGCTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA hSIRPα-VαC1βC2α 58MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVA (amino acid sequence)AGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAVRATPEHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPAGDSVSYSIHSTARVVLTRGDVHSQVICEIAHITLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK hSIRPα-VαC1αC2β 59ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT (nucleotide sequence)GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGAAGAGGAACTGCAAGTGATCCAGCCCGACAAGTCCGTGCTGGTGGCTGCTGGCGAGACTGCCACCCTGAGATGTACCGCCACCTCCCTGATCCCCGTGGGCCCTATCCAGTGGTTTAGAGGCGCTGGCCCTGGCAGAGAGCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACCGTGTCCGACCTGACCAAGCGGAACAACATGGACTTCTCCATCCGGATCGGCAACATCACCCCTGCCGATGCCGGCACCTACTACTGCGTGAAGTTCCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCTGGCACCGAGCTGTCTGTGCGGGCTAAACCTTCTGCCCCTGTGGTGTCTGGCCCTGCCGCTAGAGCTACCCCTCAGCACACCGTGTCTTTTACCTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGGTTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACCCTGTGGGCGAGTCCGTGTCCTACTCCATCCACTCCACCGCCAAGGTGGTGCTGACACGCGAGGACGTGCACTCCCAAGTGATCTGCGAGGTGGCCCACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAACCTGTCCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCCTATGAGAGCCGAGAACCAGGCCAACGTGACCTGCCAGGTGTCCAACTTCTACCCTCGGGGCCTGCAGCTGACCTGGCTGGAAAACGGCAATGTGTCCCGGACCGAGACAGCCTCCACCCTGATCGAGAACAAGGATGGCACCTACAATTGGATGTCCTGGCTGCTCGTGAACACCTGTGCCCACCGGGACGATGTGGTGCTGACCTGTCAGGTGGAACACGATGGCCAGCAGGCCGTGTCCAAGTCCTACGCTCTGGAAGTGTCCGCCCACCCCAAAGAGCAGGGCTCTAATACCGCCGCTGAGAACACCGGCTCCAACGAGCGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGGTGGCACTGCTGATGGCCGCTCTGTACCTCGTGCGGATCCGGCAGAAGAAGGCTCAGGGCTCCACCTCCTCCACCAGACTGCACGAGCCTGAGAAGAACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACGCCGACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCTGCCGAGCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCTCAGCCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGGTGCACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCCTAGCTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA hSIRPα-VαC1αC2β 60MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVA (amino acid sequence)AGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPMRAENQANVTCQVSNFYPRGLQLTWLENGNVSRTETASTLIENKDGTYNWMSWLLVNTCAHRDDVVLTCQVEHDGQQAVSKSYALEVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human 61ATGGAGCCCGCCGGCCCGGCCCCCGGCCGCCTCGGGCCGCTGCTCT SIRPαV1 (P74A)GCCTGCTGCTCGCCGCGTCCTGCGCCTGGTCAGGAGTGGCGGGTGA (nucleotide sequence)GGAGGAGCTGCAGGTGATTCAGCCTGACAAGTCCGTGTTGGTTGCAGCTGGAGAGACAGCCACTCTGCGCTGCACTGCGACCTCTCTGATCCCTGTGGGGCCCATCCAGTGGTTCAGAGGAGCTGGA:CAGGCCGGGAATTAATCTACAATCAAAAAGAAGGCCACTTCCCCCGGGTAACAACTGTTTCAGACCTCACAAAGAGAAACAACATGGACTTTTCCATCCGCATCGGTAACATCACCCCAGCAGATGCCGGCACCTACTACTGTGTGAAGTTCCGGAAAGGGAGCCCCGATGACGTGGAGTTTAAGTCTGGAGCAGGCACTGAGCTGTCTGTGCGCGCCAAACCCTCTGCCCCCGTGGTATCGGGCCCTGCGGCGAGGGCCACACCTCAGCACACAGTGAGCTTCACCTGCGAGTCCCACGGCTTCTCACCCAGAGACATCACCCTGAAATGGTTCAAAAATGGGAATGAGCTCTCAGACTTCCAGACCAACGTGGACCCCGTAGGAGAGAGCGTGTCCTACAGCATCCACAGCACAGCCAAGGTGGTGCTGACCCGCGAGGACGTTCACTCTCAAGTCATCTGCGAGGTGGCCCACGTCACCTTGCAGGGGGACCCTCTTCGTGGGACTGCCAACTTGTCTGAGACCATCCGAGTTCCACCCACCTTGGAGGTTACTCAACAGCCCGTGAGGGCAGAGAACCAGGTGAATGTCACCTGCCAGGTGAGGAAGTTCTACCCCCAGAGACTACAGCTGACCTGGTTGGAGAATGGAAACGTGTCCCGGACAGAAACGGCCTCAACCGTTACAGAGAACAAGGATGGTACCTACAACTGGATGAGCTGGCTCCTGGTGAATGTATCTGCCCACAGGGATGATGTGAAGCTCACCTGCCAGGTGGAGCATGACGGGCAGCCAGCGGTCAGCAAAAGCCATGACCTGAAGGTCTCAGCCCACCCGAAGGAGCAGGGCTCAAATACCGCCGCTGAGAACACTGGATCTAATGAACGGAACATCTATATTGTGGTGGGTGTGGTGTGCACCTTGCTGGTGGCCCTACTGATGGCGGCCCTCTACCTCGTCCGAATCAGACAGAAGAAAGCCCAGGGCTCCACTTCTTCTACAAGGTTGCATGAGCCCGAGAAGAATGCCAGAGAAATAACACAGGACACAAATGATATCACATATGCAGACCTGAACCTGCCCAAGGGGAAGAAGCCTGCTCCCCAGGCTGCGGAGCCCAACAACCACACGGAGTATGCCAGCATTCAGACCAGCCCGCAGCCCGCGTCGGAGGACACCCTCACCTATGCTGACCTGGACATGGTCCACCTCAACCGGACCCCCAAGCAGCCGGCCCCCAAGCCTGAGCCGTCCTTCTCAGAGTACGCCAGCGTCCAGGTCCCGAGGAAG human 62MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVA SIRPαV1 (P74A)AGETATLRCTATSLIPVGPIQWFRGAGAGRELIYNQKEGHFPRVTT (amino acid sequence)VSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human kappa constant 63CGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACG domain (nucleotideAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAA sequence)CTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGC human kappa constant 64RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA domain (proteinLQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ sequence) GLSSPVTKSFNRGEChuman IgG4 constant 65 GCTTCCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTGCTCCAdomains (including GATCCACCTCCGAGTCTACCGCCGCTCTGGGCTGCCTCGTGAAGGA S228P)CTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTG (nucleotide sequence)ACCTCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCAGCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCCTCCAACACCAAGGTGGACAAGCGGGTGGAATCTAAGTACGGCCCTCCCTGCCCTCCTTGCCCAGCCCCTGAATTTCTGGGCGGACCTTCTGTGTTTCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAGGAAGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCTCCAAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCCTTCTTTCTGTACTCTCGCCTGACCGTGGACAAGTCCCGGTGGCAGGAAGGCAACGTGTTCTCCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCC CTGTCTCTGGGAAAAhuman IgG4 constant 66 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALdomains (including TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT S228P)KVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPE (protein sequence)VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLGK human IgG2 constant67 GCTTCTACAAAGGGCCCCAGCGTGTTCCCTCTGGCTCCTTGTAGCA domains (nucleotideGAAGCACCAGCGAGTCTACAGCCGCTCTGGGCTGTCTGGTCAAGGA sequence)CTACTTTCCCGAGCCTGTGACCGTGTCCTGGAATAGCGGAGCACTGACAAGCGGCGTGCACACCTTTCCAGCTGTGCTGCAAAGCTCCGGCCTGTACTCTCTGTCCAGCGTGGTCACAGTGCCCAGCAGCAATTTTGGCACCCAGACCTACACCTGTAATGTGGACCACAAGCCTAGCAACACCAAGGTGGACAAGACCGTGGAACGGAAGTGCTGCGTGGAATGCCCTCCTTGTCCTGCTCCTCCAGTGGCTGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACAGCACCTTCAGAGTGGTGTCCGTGCTGACCGTGGTGCATCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGACCAAAGGCCAGCCTCGCGAGCCCCAGGTTTACACACTTCCTCCAAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCCAGCGACATCX₁CCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTATGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCT GAGCCCCGGCAAA wherein:X₁ = G, T human IgG2 constant 68ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL domains (proteinTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNT sequence)KVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIX₁VEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK wherein: X₁ = A, S40A heavy chain 69 SYWMH CDR1 (amino acid sequence) 40A heavy chain 70AIYPVNNDTTYNQKFKG CDR2 (amino acid sequence) 40A heavy chain 71SFYYSLDAAWFVY CDR3 (amino acid sequence) 40A light chain CDR1 72RASQDIGSRLN (amino acid sequence) 40A light chain CDR2 73 ATSSLDS(amino acid sequence) 40A light chain CDR3 74 LQYASSPFT(amino acid sequence) humanized 40 heavy 75EVQX₁X₂QSGAX₃X₄X₅KPGASVKX₆SCKASGSTFTSYWMHWVS₇QX₈ chain variable regionPGQGLEWX₉GAIYPVNSDTTYNQKFKGX₁₀X₁₁TX₁₂TVX₁₃X₁₄SX₁₅S (consensus sequence)TX₁₆YMX₁₇LSSLX₁₈X₁₉EDX₂₀AVYYCX₂₁RSFYYSLDAAWFVYWGQG TX₂₂X₂₃TVSS wherein:X₁ = F, L X₂ = Q, R, V X₃ = E, V X₄ = L, V X₅ = A, K, V X₆ = L, M, VX₇ = K, R X₈ = A, R, T X₉ = I, M X₁₀ = K, R X₁₁ = A, V X₁₂ = L, MX₁₃ = D, V X₁₄ = K, T X₁₅ = A, S, T X₁₆ = A, V X₁₇ = E, Q X₁₈ = R, TX₁₉ = F, S X₂₀ = S, T X₂₁ = A, T X₂₂ - L, T X₂₃ = L, Vhumanized 40 light 76 DIQMTQSPSSLSASX₁GX₂RVX₃ITCRASQDIGSRLNWLQQX₄PGKAchain variable region X₅KRLIYATSSLDSGVPX₆RFSGSX₇SGX₈X₉X₁₀X₁₁LTISX₁₂LQPE(consensus sequence) DFATYYCLQYASSPFTFGX₁₃GTKX₁₄EIX₁₅ wherein: X₁ = L, VX₂ = D, E X₃ = S, T X₄ = K, T X₅ = I, P X₆ = K, S X₇ = G, R X₈ = S, TX₉ = D, E X₁₀ = F, Y X₁₁ = S, T X₁₂ = G, S X₁₃ = G, Q X₁₄ = L, VX₁₅ = H, K hSIRPα.40AVH1 77GAGGTGCAGTTCTTGCAGTCTGGTGCCGTGCTGGCTAGACCTGGAA (nucleotide sequence)CCTCCGTGAAGATCTCCTGCAAGGCCTCCGGCTCCACCTTCACCTCTTACTGGATGCACTGGGTCAAGCAGAGGCCTGGACAGGGACTCGAATGGATCGGCGCTCTGTACCCTGTGAACTCCGACACCACCTACAACCAGAAGTTCAAGGGCAGAGCCAAGCTGACCGTGGCCACCTCTGCTTCTATCGCCTACCTGGAATTTTCCAGCCTGACCAACGAGGACTCCGCCGTGTACTACTGCGCCCGGTCCTTCTACTACTCTCTGGACGCCGCTTGGTTTGTGTACTGGGGCCAGGGAACTCTGGTGACCGTGTCCTCT hSIRPα.40AVH1 78EVQFLQSGAVLARPGTSVKISCKASGSTFTSYWMHWVKQRPGQGLE (amino acid sequence)WIGALYPVNSDTTYNQKFKGRAKLTVATSASIAYLEFSSLTNEDSAVYYCARSFYYSLDAAWFVYWGQGTLVTVSS hSIRPα.40AVH2 79GAGGTGCAGCTGGTTCAGTCTGGCGCTGAGGTTGTGAAGCCTGGCG (nucleotide sequence)CTTCCGTGAAGCTGTCCTGCAAGGCTTCTGGCTCCACCTTCACCAGCTACTGGATGCACTGGGTCAAGCAGGCCCCTGGACAAGGCCTGGAATGGATCGGCGCTATCTACCCCGTGAACTCCGACACCACCTACAACCAGAAGTTCAAGGGCAAAGCTACCCTGACCGTGGACAAGTCTGCCTCCACCGCCTACATGGAACTGTCCAGCCTGAGATCTGAGGACACCGCCGTGTACTACTGCACCCGGTCCTTCTACTACTCCCTGGACGCCGCTTGGTTTGTGTATTGGGGCCAGGGAACACTGGTGACCGTGTCCTCT hSIRPα.40AVH2 80EVQLVQSGAEVVKPGASVKLSCKASGSTFTSYWMHWVKQAPGQGLE (amino acid sequence)WIGAIYPVNSDTTYNQKFKGKATLTVDKSASTAYMELSSLRSEDTAVYYCTRSFYYSLDAAWFVYWGQGTLVTVSS hSIRPα.40AVH3 81GAGGTGCAGCTGAGACAGTCTGGCGCTGTGCTTGTGAAGCCTGGCG (nucleotide sequence)CCTCCGTGAAGATGTCCTGCAAGGCTTCTGGCTCCACCTTCACCAGCTACTGGATGCACTGGGTCAAGCAGACCCCTGGACAGGGACTCGAGTGGATCGGCGCTATCTACCCTGTGAACTCCGACACCACCTACAACCAGAAGTTCAAGGGCAAAGCTACCCTGACCGTGGACAAGTCCTCCTCCACCGCTTACATGCAGCTGTCCAGCCTGACCTCTGAGGACTCCGCCGTGTACTACTGCGCCCGGTCCTTCTACTACTCTCTGGACGCCGCTTGGTTTGTGTACTGGGGCCAGGGCACAACCCTGACAGTGTCCTCT hSIRPα.40AVH3 82EVQLRQSGAVLVKPGASVKMSCKASGSTFTSYWMHWVKQTPGQGLE (amino acid sequence)WIGAIYPVNSDTTYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARSFYYSLDAAWFVYWGQGTTLTVSS hSIRPα.40AVH4 83GAGGTGCAGTTCGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCG (nucleotide sequence)CCTCTGTGAAGGTGTCCTGCAAGGCTTCTGGCTCCACCTTCACCAGCTACTGGATGCACTGGGTCCGACAGGCTCCAGGACAAGGCTTGGAATGGATGGGCGCTATCTACCCCGTGAACTCCGACACCACCTACAACCAGAAATTCAAGGGCAGAGTGACCATGACCGTCGTGACCTCCACCTCCACCGTGTACATGGAACTGTCCAGCCTGAGATCCGAGGACACCGCCGTGTACTACTGCGCCCGGTCCTTCTACTACTCTCTGGACGCCGCTTGGTTTGTGTACTGGGGCCAGGGAACTCTGGTGACCGTGTCCTCT hSIRPα.40AVH4 84EVQFVQSGAEVKKPGASVKVSCKASGSTFTSYWMHWVRQAPGQGLE (amino acid sequence)WMGAIYPVNSDTTYNQKFKGRVTMTVVTSTSTVYMELSSLRSEDTAVYYCARSFYYSLDAAWFVYWGQGTLVTVSS hSIRPα.40AVH5 85GAGGTCCAGCTGCAACAGTCTGGTGCCGTGTTGGCTAAGCCTGGCG (nucleotide sequence)CCTCCGTGAAGATGTCCTGCAAGGCTTCTGGCTCCACCTTCACCAGCTACTGGATGCACTGGGTCAAGCAGAGGCCTGGACAGGGACTCGAGTGGATCGGCGCTATCTACCCTGTGAACTCCGACACCACCTACAACCAGAAGTTCAAGGGCAAAGCTACCCTGACCGTGGACAAGTCCTCCTCCACCGCTTACATGCAGCTGTCCAGCCTGACCTTCGAGGACTCCGCCGTGTACTACTGCGCCCGGTCCTTCTACTACTCTCTGGACGCCGCTTGGTTTGTGTACTGGGGCCAGGGCACAACCCTGACAGTGTCCTCT hSIRPα.40AVH5 86EVQLQQSGAVLAKPGASVKMSCKASGSTFTSYWMHWVKQRPGQGLE (amino acid sequence)WIGAIYPVNSDTTYNQKFKGKATLTVDKSSSTAYMQLSSLTFEDSAVYYCARSFYYSLDAAWFVYWGQGTTLTVSS hSIRPα.40AVH6 87GAGGTGCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCG (nucleotide sequence)CCTCTGTGAAGGTGTCCTGCAAGGCTTCTGGCTCCACCTTCACCAGCTACTGGATGCACTGGGTCCGACAGGCTCCAGGACAAGGCTTGGAATGGATGGGCGCTATCTACCCCGTGAACTCCGACACCACCTACAACCAGAAATTCAAGGGCAGAGTGACCATGACCGTGGACACCTCCACCAGCACCGTGTACATGGAACTGTCCAGCCTGAGATCCGAGGACACCGCCGTGTACTACTGCGCCCGGTCCTTCTACTACTCTCTGGACGCCGCTTGGTTTGTGTACTGGGGCCAGGGAACTCTGGTGACCGTGTCCTCT hSIRPα.40AVH6 88EVQLVQSGAEVKKPGASVKVSCKASGSTFTSYWMHWVRQAPGQGLE (amino acid sequence)WMGAIYPVNSDTTYNQKFKGRVTMTVDTSTSTVYMELSSLRSEDTAVYYCARSFYYSLDAAWFVYWGQGTLVTVSS hSIRPα.40AVL1 89GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGG (nucleotide sequence)GCGACAGAGTGACCATCACCTGTAGAGCCTCTCAGGACATCGGCTCCAGACTGAACTGGCTGCAGCAGACCCCTGGCAAGGCCATCAAGAGACTGATCTACGCCACCTCCAGCCTGGATTCTGGCGTGCCCTCTAGATTCTCCGGCTCTAGATCTGGCACCGACTTCTCCCTGACCATCTCTGGACTGCAGCCTGAGGACTTCGCCACCTACTACTGTCTGCAGTACGCCAGCTCTCCATTCACCTTTGGCGGAGGCACCAAGGTGGAAATCCAC hSIRPα.40AVL1 90DIQMTQSPSSLSASVGDRVTITCRASQDIGSRLNWLQQTPGKAIKR (amino acid sequence)LIYATSSLDSGVPSRFSGSRSGTDFSLTISGLQPEDFATYYCLQYA SSPFTFGGGTKVEIHhSIRPα.40AVL2 91 GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGG(nucleotide sequence) GCGACAGAGTGACCATCACCTGTAGAGCCTCTCAGGACATCGGCTCCAGACTGAACTGGCTGCAGCAGAAGCCTGGCAAGGCCATCAAGAGACTGATCTACGCCACCTCCAGCCTGGATTCTGGCGTGCCCTCTAGATTCTCCGGCTCTAGATCTGGCACCGACTTTACCCTGACAATCAGCTCCCTGCAGCCTGAGGACTTCGCCACCTACTACTGTCTGCAGTACGCCTCCTCTCCATTCACCTTTGGCCAGGGCACCAAGGTGGAAATCAAG hSIRPα.40AVL2 92DIQMTQSPSSLSASVGDRVTITCRASQDIGSRLNWLQQKPGKAIKR (amino acid sequence)LIYATSSLDSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCLQYA SSPFTFGQGTKVEIKhSIRPα.40AVL3 93 GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGG(nucleotide sequence) GCGACAGAGTGACCATCACCTGTAGAGCCTCTCAGGACATCGGCTCCAGACTGAACTGGCTGCAGCAGAAGCCTGGCAAGGCCATCAAGAGACTGATCTACGCCACCTCCAGCCTGGATTCTGGCGTGCCCTCTAGATTCTCCGGCTCTAGATCTGGCACCGACTTTACCCTGACAATCAGCTCCCTGCAGCCTGAGGACTTCGCCACCTACTACTGTCTGCAGTACGCCAGCTCTCCATTCACCTTTGGCGGAGGCACCAAGCTGGAAATCAAG hSIRPα.40AVL3 94DIQMTQSPSSLSASVGDRVTITCRASQDIGSRLNWLQQKPGKAIKR (amino acid sequence)LIYATSSLDSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCLQYA SSPFTFGGGTKLEIKhSIRPα.40AVL4 95 GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGG(nucleotide sequence) GCGACAGAGTGACCATCACCTGTAGAGCCTCTCAGGACATCGGCTCCAGACTGAACTGGCTGCAGCAGAAGCCTGGCAAGGCCCCTAAGAGACTGATCTACGCCACCTCCAGCCTGGATTCTGGCGTGCCCTCTAGATTCTCCGGCTCTGGCTCTGGCACCGAGTTTACCCTGACAATCAGCTCCCTGCAGCCTGAGGACTTCGCCACCTACTACTGTCTGCAGTACGCCAGCTCTCCATTCACCTTTGGCGGAGGCACCAAGGTGGAAATCAAG hSIRPα.40AVL4 96DIQMTQSPSSLSASVGDRVTITCRASQDIGSRLNWLQQKPGKAPKR (amino acid sequence)LIYATSSLDSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYA SSPFTFGGGTKVEIKhSIRPα.40AVL5 97 GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGG(nucleotide sequence) GCGACAGAGTGACCATCACCTGTAGAGCCTCTCAGGACATCGGCTCCAGACTGAACTGGCTGCAGCAGAAGCCTGGCAAGGCCATCAAGAGACTGATCTACGCCACCTCCAGCCTGGATTCTGGCGTGCCCAAGAGATTCTCCGGCTCTAGATCCGGCTCCGACTATACCCTGACAATCAGCTCCCTGCAGCCTGAGGACTTCGCCACCTACTACTGTCTGCAGTACGCCTCCTCTCCATTCACCTTTGGCCAGGGCACCAAGGTGGAAATCAAG hSIRPα.40AVL5 98DIQMTQSPSSLSASVGDRVTITCRASQDIGSRLNWLQQKPGKAIKR (amino acid sequence)LIYATSSLDSGVPKRFSGSRSGSDYTLTISSLQPEDFATYYCLQYA SSPFTFGQGTKVEIKhSIRPα.40AVL6 99 GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCTGCTTCCCTGG(nucleotide sequence) GCGAGAGAGTGTCCATCACCTGTAGAGCCTCTCAGGACATCGGCTCCAGACTGAACTGGCTGCAGCAGAAGCCTGGCAAGGCCATCAAGAGACTGATCTACGCCACCTCCAGCCTGGATTCTGGCGTGCCCTCTAGATTCTCCGGCTCTAGATCTGGCACCGACTTTACCCTGACAATCAGCTCCCTGCAGCCTGAGGACTTCGCCACCTACTACTGTCTGCAGTACGCCAGCTCTCCATTCACCTTTGGCGGAGGCACCAAGGTGGAAATCAAG hSIRPα.40AVL6 100DIQMTQSPSSLSASLGERVSITCRASQDIGSRLNWLQQKPGKAIKR (amino acid sequence)LIYATSSLDSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCLQYA SSPFTFGGGTKVEIKhSIRPα.40A mouse 101 GAGGTTCAGTTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCAGGGAVH (nucleotide CTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTCCACCTTTACCAG sequence)CTACTGGATGCACTGGGTAAAACAGGGGCCTGGACAGGGTCTGCAATGGATTGGCGCTATTTATCCTGTAAATAATGATACTACCTATAATCAGAAGTTCAAGGGCAAGGCCGAACTCACTGTAGTCACTTCCACCAGCACTGCCTACATGGAGGTCAGTAGTCTGACAAATGAGGACTCTGCGGTCTATTACTGTACAAGATCGTTCTACTATAGTCTCGACGCGGCCTGGTTTGTTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA hSIRPα.40A mouse 102EVQFQQSGTVLARPGTSVKMSCKASGSTFTSYWMHWVKQGPGQGLQ VH (amino acidWIGAIYPVNNDTTYNQKFKGKAELTVVTSTSTAYMEVSSLTNEDSA sequence)VYYCTRSFYYSLDAAWFVYWGQGTLVTVSA hSIRPα.40A mouse 103GACATCCAGATGACCCAGTCTCCATCCTCCTTATCTGCCTCTCTGG VL (nucleotideGAGAAAGAGTCAGTCTCACTTGTCGGGCAAGTCAGGACATTGGTAG sequence)TAGGTTAAACTGGCTTCAGCAGGAACCAGATGGAACTATTAAACGCCTGATCTACGCCACATCCAGTTTAGATTCTGGTGTCCCCAAAAGGTTCAGTGGCAGTAGGTCTGGGTCAGATTATTCTCTCACCATCAGCGGCCTTGAGTCTGAAGACTTTGTAGACTATTACTGTCTACAATATGCTAGTTCTCCGTTCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAC hSIRPα.40A mouse 104DIQMTQSPSSLSASLGERVSLTCRASQDIGSRLNWLQQEPDGTIKR VL (amino acidLIYATSSLDSGVPKRFSGSRSGSDYSLTISGLESEDFVDYYCLQYA sequence) SSPFTFGGGTKLEINhSIRPα.40A mouse 105 EVQFQQSGTVLARPGTSVKMSCKASGSTFTSYWMHWVKQGPGQGLQheavy chain (amino WIGAIYPVNNDTTYNQKFKGKAELTVVTSTSTAYMEVSSLTNEDSAacid sequence; VYYCTRSFYYSLDAAWFVYWGQGTLVTVSAAKTTPPSVYPLAPGSAconstant domain AQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYunderlined, signal TLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCIpeptide not shown) CTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK hSIRPα.40A mouse 106DIQMTQSPSSLSASLGERVSLTCRASQDIGSRLNWLQQEPDGTIKR light chain (amino acidLIYATSSLDSGVPKRFSGSRSGSDYSLTISGLESEDFVDYYCLQYA sequence; constantSSPFTFGGGTKLEINRADAAPTVSIFPPSSEQLTSGGASVVCFLNN domain underlined,FYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKD signal peptide notEYERHNSYTCEATHKTSTSPIVKSFNRNEC shown) rhSIRPα/Fc (amino 107(GVAG)EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFR acid sequence)GAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK rhSIRPγ/Fc (amino 108VLWFRGVGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRISSI acid sequence)TPADVGTYYCVKFRKGSPENVEFKSGPGTEMALGAKPSAPVVLGPAARTTPEHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPTGQSVAYSIRSTARVVLDPWDVRSQVICEVAHVTLQGDPLRGTANLSEAIRVPPTLEVTQQPMRAGNQVNVTCQVRKFYPQSLQLTWLENGNVCQRETASTLTENKDGTYNWTSWFLVNISDQRDDVVLTCQVKHDGQLAVSKRLALEVTVHQKDQSSDATPGPASIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK rhCD47/Fc (amino 109QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRD acid sequence)IYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hSIRP-VγC1βC2β 110ATGCCCGTGCCTGCCTCTTGGCCTCATCTGCCCAGCCCCTTTCTGC (nucleotide sequence)TGATGACCCTGCTGCTGGGCAGGCTGACAGGCGTGGCAGGCGAAGAGGAACTGCAGATGATCCAGCCCGAGAAGCTGCTGCTCGTGACCGTGGGCAAGACCGCCACCCTGCACTGCACCGTGACATCCCTGCTGCCTGTGGGACCCGTGCTGTGGTTTAGAGGCGTGGGCCCTGGCAGAGAGCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACCGTGTCCGACCTGACCAAGCGGAACAACATGGACTTCTCCATCCGGATCTCCAGCATCACCCCTGCCGACGTGGGCACCTACTACTGCGTGAAGTTCCGGAAGGGCTCCCCCGAGAACGTGGAGTTCAAGTCTGGCCCAGGCACCGAGATGGCCCTGGGCGCTAAACCTTCTGCCCCTGTGGTGTCTGGACCTGCCGTGCGGGCTACCCCTGAGCACACCGTGTCTTTTACCTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGGTTCAAGAACGGCAACGAGCTGTCCGACTTCCAGACCAACGTGGACCCTGCCGGCGACTCCGTGTCCTACTCCATCCACTCTACCGCCAGAGTGGTGCTGACCAGAGGCGACGTGCACTCCCAAGTGATCTGCGAGATCGCCCATATCACACTGCAGGGCGACCCCCTGAGAGGCACCGCCAATCTGTCTGAGGCCATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCCTATGAGAGCCGAGAACCAGGCCAACGTGACCTGTCAGGTGTCCAACTTCTACCCTCGGGGCCTGCAGCTGACCTGGCTGGAAAACGGCAATGTGTCCCGGACCGAGACAGCCTCCACCCTGATCGAGAACAAGGACGGCACCTACAATTGGATGTCCTGGCTGCTCGTGAACACCTGTGCCCACAGGGACGACGTGGTGCTGACATGCCAGGTGGAACACGATGGCCAGCAGGCCGTGTCCAAGTCCTACGCCCTGGAAATCTCCGCCCATCAGAAAGAGCACGGCTCCGATATCACCCACGAGGCCGCTCTGGCTCCTACCGCTCCTCTGCTGGTGGCTCTGCTGCTGGGACCTAAGCTGCTGCTGGTCGTGGGCGTGTCCGCCATCTACATCTGCTGGAAGCAGAAGGCCTG A hSIRP-VγC1βC2β 111MPVPASWPHLPSPFLLMTLLLGRLTGVAGEEELQMIQPEKLLLVTV (amino acid sequence)GKTATLHCTVTSLLPVGPVLWFRGVGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPENVEFKSGPGTEMALGAKPSAPVVSGPAVRATPEHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPAGDSVSYSIHSTARVVLTRGDVHSQVICEIAHITLQGDPLRGTANLSEAIRVPPTLEVTQQPMRAENQANVTCQVSNFYPRGLQLTWLENGNVSRTETASTLIENKDGTYNWMSWLLVNTCAHRDDVVLTCQVEHDGQQAVSKSYALEISAHQKEHGSDITHEAALAPTAPLLVALLLGPKLLLVVGVSAIYICWKQKA hSIRP-VβC1γC2β 112ATGCCCGTGCCTGCCTCTTGGCCTCATCTGCCCAGCCCCTTTCTGC (nucleotide sequence)TGATGACCCTGCTGCTGGGCAGGCTGACAGGCGTGGCAGGCGAAGATGAGCTGCAAGTGATCCAGCCCGAGAAGTCCGTGTCTGTGGCCGCTGGCGAGTCTGCCACCCTGAGATGCGCTATGACCTCCCTGATCCCCGTGGGCCCCATCATGTGGTTTAGAGGCGCTGGCGCTGGCAGAGAGCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACCGTGTCCGAGCTGACCAAGCGGAACAACCTGGACTTCTCCATCTCCATCAGCAACATCACCCCTGCCGACGCCGGCACCTACTACTGCGTGAAGTTCCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCTGGAACCGAGCTGTCCGTGCGGGCTAAACCTTCTGCCCCTGTGGTGCTGGGACCTGCCGCTAGAACCACCCCTGAGCACACCGTGTCTTTTACCTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGGTTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACCCTACCGGCCAGTCCGTGGCCTACTCCATCAGATCCACCGCCAGAGTGGTGCTGGACCCTTGGGATGTGCGGTCCCAAGTGATCTGCGAGGTGGCCCATGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAATCTGTCTGAGGCCATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCCTATGAGAGCCGAGAACCAGGCCAACGTGACCTGCCAGGTGTCCAACTTCTACCCTCGGGGCCTGCAGCTGACCTGGCTGGAAAACGGCAATGTGTCCCGGACCGAGACAGCCTCCACCCTGATCGAGAACAAGGATGGCACCTACAATTGGATGTCCTGGCTGCTCGTGAACACCTGTGCCCACCGGGATGACGTGGTGCTGACTTGTCAGGTGGAACACGACGGCCAGCAGGCCGTGTCCAAGTCCTACGCCCTGGAAATCTCCGCCCATCAGAAAGAGCACGGCTCCGATATCACCCACGAGGCCGCTCTGGCTCCTACCGCTCCTCTGCTGGTGGCTCTGCTGCTGGGACCTAAGCTGCTGCTGGTCGTGGGCGTGTCCGCCATCTACATCTGCTGGAAGCAGAAGGCCTG A hSIRP-VβC1γC2β 113MPVPASWPHLPSPFLLMTLLLGRLTGVAGEDELQVIQPEKSVSVAA (amino acid sequence)GESATLRCAMTSLIPVGPIMWFRGAGAGRELIYNQKEGHFPRVTTVSELTKRNNLDFSISISNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVLGPAARTTPEHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPTGQSVAYSIRSTARVVLDPWDVRSQVICEVAHVTLQGDPLRGTANLSEAIRVPPTLEVTQQPMRAENQANVTCQVSNFYPRGLQLTWLENGNVSRTETASTLIENKDGTYNWMSWLLVNTCAHRDDVVLTCQVEHDGQQAVSKSYALEISAHQKEHGSDITHEAALAPTAPLLVALLLGPKLLLVVGVSAIYICWKQKA hSIRP-VβC1βC2γ 114ATGCCCGTGCCTGCCTCTTGGCCTCATCTGCCCAGCCCCTTTCTGC (nucleotide sequence)TGATGACCCTGCTGCTGGGCAGGCTGACAGGCGTGGCAGGCGAAGATGAGCTGCAAGTGATCCAGCCCGAGAAGTCCGTGTCTGTGGCCGCTGGCGAGTCTGCCACCCTGAGATGCGCTATGACCTCCCTGATCCCCGTGGGCCCCATCATGTGGTTTAGAGGCGCTGGCGCTGGCAGAGAGCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACCGTGTCCGAGCTGACCAAGCGGAACAACCTGGACTTCTCCATCTCCATCAGCAACATCACCCCTGCCGACGCCGGCACCTACTACTGCGTGAAGTTCCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCTGGAACCGAGCTGTCCGTGCGGGCTAAACCTTCTGCCCCTGTGGTGTCTGGACCTGCTGTGCGCGCTACCCCTGAGCACACCGTGTCTTTTACCTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGGTTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACCCTGCCGGCGACTCCGTGTCCTACTCCATCCACTCTACCGCCAGAGTGGTGCTGACCAGAGGCGACGTGCACTCCCAAGTGATCTGCGAGATCGCCCATATCACACTGCAGGGCGACCCCCTGAGAGGCACCGCCAATCTGTCTGAGGCCATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCCTATGAGAGTGGGCAACCAAGTGAACGTGACCTGCCAAGTGCGGAAGTTCTACCCCCAGTCCCTGCAGCTGACTTGGAGCGAGAATGGCAACGTGTGCCAGAGAGAGACAGCCTCCACCCTGACCGAGAACAAGGACGGAACCTACAACTGGACCTCCTGGTTCCTCGTGAACATCTCCGACCAGCGGGACGACGTGGTGCTGACATGCCAAGTGAAGCACGATGGACAGCTGGCCGTGTCCAAGCGGCTGGCTCTGGAAGTGACAGTGCACCAGAAAGAGCACGGCTCCGACATCACCCACGAGGCCGCTCTGGCTCCTACAGCTCCTCTGCTGGTGGCTCTGCTGCTGGGACCTAAGCTGCTGCTGGTCGTGGGCGTGTCCGCCATCTACATCTGCTGGAAGCAGAAGGCCTG A hSIRP-VβC1βC2γ 115MPVPASWPHLPSPFLLMTLLLGRLTGVAGEDELQVIQPEKSVSVAA (amino acid sequence)GESATLRCAMTSLIPVGPIMWFRGAGAGRELIYNQKEGHFPRVTTVSELTKRNNLDFSISISNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAVRATPEHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPAGDSVSYSIHSTARVVLTRGDVHSQVICEIAHITLQGDPLRGTANLSEAIRVPPTLEVTQQPMRVGNQVNVTCQVRKFYPQSLQLTWSENGNVCQRETASTLTENKDGTYNWTSWFLVNISDQRDDVVLTCQVKHDGQLAVSKRLALEVTVHQKEHGSDITHEAALAPTAPLLVALLLGPKLLLVVGVSAIYICWKQKA human SIRPβL 116ATGCCTGTGCCTGCCTCTTGGCCTCATCTGCCCTCTCCATTTCTGC (nucleotide sequence)TGATGACCCTGCTGCTGGGCAGACTGACAGGTGTTGCTGGCGAAGAGGAACTGCAAGTGATCCAGCCTGACAAGAGCATCTCTGTGGCCGCTGGCGAATCTGCCACACTGCACTGTACCGTGACATCTCTGATCCCTGTGGGCCCCATCCAGTGGTTTAGAGGTGCTGGACCTGGCAGAGAGCTGATCTACAACCAGAAAGAGGGACACTTCCCCAGAGTGACCACCGTGTCCGACCTGACCAAGCGGAACAACATGGACTTCAGCATCCGGATCAGCAACATCACCCCTGCCGATGCCGGCACCTACTACTGCGTGAAGTTCAGAAAGGGCAGCCCCGACCACGTCGAGTTTAAAAGCGGAGCCGGCACAGAGCTGAGCGTGCGGGCTAAACCTTCTGCTCCTGTGGTGTCTGGACCAGCCGCTAGAGCTACACCTCAGCACACCGTGTCTTTTACCTGCGAGAGCCACGGCTTCAGCCCCAGAGATATCACCCTGAAGTGGTTCAAGAACGGCAACGAGCTGTCCGACTTCCAGACCAATGTGGACCCAGCCGGCGATAGCGTGTCCTACAGCATTCACAGCACCGCCAAGGTGGTGCTGACCCGGGAAGATGTGCACAGCCAAGTGATTTGCGAGGTGGCCCACGTTACCCTGCAAGGCGATCCTCTGAGAGGAACCGCCAACCTGAGCGAGACAATCCGGGTGCCACCTACACTGGAAGTGACCCAGCAGCCTGTGCGGGCCGAGAATCAAGTGAACGTGACCTGCCAAGTGCGGAAGTTCTACCCTCAGAGACTGCAGCTGACCTGGCTGGAAAACGGCAATGTGTCCCGGACCGAGACAGCCAGCACACTGACCGAGAACAAGGATGGCACCTACAATTGGATGAGCTGGCTGCTGGTCAATGTGTCTGCCCACCGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGATGGCCAGCCTGCCGTGTCTAAGAGCCACGACCTGAAGGTGTCCGCTCATCCCAAAGAGCAGGGCAGCAATACTGCCCCTGGACCTGCTCTTGCTTCTGCCGCTCCTCTGCTGATCGCCTTTCTGCTGGGACCTAAGGTGCTGCTGGTTGTGGGAGTGTCCGTGATCTACGTGTACTGGAAGCAGAAGGCC human SIRPβL (amino 117MPVPASWPHLPSPFLLMTLLLGRLTGVAGEEELQVIQPDKSISVAA acid sequence)GESATLHCTVTSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDHVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPAGDSVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTLTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAPGPALASAAPLLIAFLLGPKVLLVVGVSVIYVYWKQKA human IgG1 constant 118GCCAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCA domains (nucleotideAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGA sequence)CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG AGCCTCTCCCTGTCTCCGGGTAAAhuman IgG1 constant 119 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALdomains (amino acid TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTsequence) KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKmouse IgG1 constant 120 AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLdomains (amino acid SSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTsequence) VDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLK NYYLKKTISRSPGKmouse kappa constant 121 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSdomain (amino acid ERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKsequence) TSTSPIVKSFNRNEC human IgG2 constant 122ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL domains, V234A-TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNT G237A-P238S-KVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEV H268A-V309L-TCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSV A330S-P331S (Sigma)LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTL mutant (amino acidPPSREEMTKNQVSLTCLVKGFYPSDIX₁VEWESNGQPENNYKTTPP sequence)MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK wherein: X₁ = A, Shuman IgG1 constant 123 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALdomains, L234A- TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTL235A mutant (amino KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRacid sequence) TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKhuman IgG1 constant 124 GCTAGCACAAAGGGCCCTAGTGTGTTTCCTCTGGCTCCCTCTTCCAdomains, L234A- AATCCACTTCTGGTGGCACTGCTGCTCTGGGATGCCTGGTGAAGGAL235A-P329G mutant TTACTTTCCTGAACCTGTGACTGTCTCATGGAACTCTGGTGCTCTG(nucleotide sequence) ACTTCTGGTGTCCACACTTTCCCTGCTGTGCTGCAGTCTAGTGGACTGTACTCTCTGTCATCTGTGGTCACTGTGCCCTCTTCATCTCTGGGAACCCAGACCTACATTTGTAATGTGAACCACAAACCATCCAACACTAAAGTGGACAAAAAAGTGGAACCCAAATCCTGTGACAAAACCCACACCTGCCCACCTTGTCCGGCGCCTGAAGCGGCGGGAGGACCTTCTGTGTTTCTGTTCCCCCCCAAACCAAAGGATACCCTGATGATCTCGCGAACCCCTGAGGTGACATGTGTGGTGGTGGATGTGTCTCATGAGGACCCCGAAGTCAAATTTAATTGGTATGTCGACGGCGTCGAGGTGCATAATGCCAAAACCAAGCCTAGAGAGGAACAGTACAATTCAACCTACAGAGTCGTCAGTGTGCTGACTGTGCTGCATCAGGATTGGCTGAATGGCAAGGAATACAAGTGTAAAGTCTCAAACAAGGCCCTGGGAGCTCCAATTGAGAAAACAATCTCAAAGGCCAAAGGACAGCCTAGGGAACCCCAGGTCTACACCCTGCCACCTTCGAGAGACGAACTGACCAAAAACCAGGTGTCCCTGACATGCCTGGTCAAAGGCTTCTACCCTTCTGACATTGCTGTGGAGTGGGAGTCAAATGGACAGCCTGAGAACAACTACAAAACAACCCCCCCTGTGCTGGATTCTGATGGCTCTTTCTTTCTGTACTCCAAACTGACTGTGGACAAGTCTAGATGGCAGCAGGGGAATGTCTTTTCTTGCTCTGTCATGCATGAGGCTCTGCATAACCACTACACTCAGAAA TCCCTGTCTCTGTCTCCCGGGAAAhuman IgG1 constant 125 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALdomains, L234A- TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTL235A-P329G mutant KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISR(amino acid sequence) TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKhuman IgG1 constant 126 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALdomains, N297Q TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTmutant (amino acid KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRsequence) TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKhuman IgG4 constant 127 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALdomains, S228P- TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTN297Q mutant (amino KVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEacid sequence) VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLGK 18D5 VH (amino acid128 QVQLQQPGAELVRPGSSVKLSCKASGYTFTSYWVHWVKQRPIQGLE sequence)WIGNIDPSDSDTHYNQKFKDKASLTVDKSSSTAYMQLSSLTFEDSAVYYCVRGGIGTMAWFAYWGQGTLVTVSA 18D5 VL (amino acid 129DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSYGNTYLYWYLQKPG sequence)QSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYF CFQGTHVPYTFGSGTKLEIKKWAR23 VH (amino 130 EVQLQQSGAELVKPGASVKLSCTASGFNIKDYYIHWVQQRTEQGLEacid sequence) WIGRIDPEDGETKYAPKFQDKATITADTSSNTAYLHLSSLTSEDTAVYYCARWGAYWGQGTLVTVSS KWAR23 VL (amino 131QIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYLYWYQQKPGSSPK acid sequence)LWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQW SSYPRTFGAGTKLELKrhSIRPα-HIS (amino 132 MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAacid sequence) AGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSN ERHHHHHH

All references cited herein are incorporated by reference to the sameextent as if each individual publication, database entry (e.g. Genbanksequences or GenelD entries), patent application, or patent, wasspecifically and individually indicated to be incorporated by reference.This statement of incorporation by reference is intended by Applicants,pursuant to 37 C.F.R. § 1.57(b)(1), to relate to each and everyindividual publication, database entry (e.g. Genbank sequences or GeneIDentries), patent application, or patent, each of which is clearlyidentified in compliance with 37 C.F.R. § 1.57(b)(2), even if suchcitation is not immediately adjacent to a dedicated statement ofincorporation by reference. The inclusion of dedicated statements ofincorporation by reference, if any, within the specification does not inany way weaken this general statement of incorporation by reference.Citation of the references herein is not intended as an admission thatthe reference is pertinent prior art, nor does it constitute anyadmission as to the contents or date of these publications or documents.To the extent that the references provide a definition for a claimedterm that conflicts with the definitions provided in the instantspecification, the definitions provided in the instant specificationshall be used to interpret the claimed invention.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. Variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1-30. (canceled)
 31. An antibody antibody having the followingcharacteristics: binds human SIRPαV1 protein having the sequence of SEQID NO: 34 with an EC₅₀<1 nM; does not cross-react with SIRPβ1 proteinhaving the sequence of SEQ ID NO: 38; and exhibits a T20 “humanness”score of at least
 79. 32. An antibody according to claim 31, wherein theantibody binds to a cell expressing human SIRPαV1 protein with anEC₅₀<10 nM; binds to a cell expressing human SIRPαV2 protein with anEC₅₀<10 nM; exhibits at least a 100-fold higher EC₅₀ for SIRPαV1(P74A)having the sequence of SEQ ID NO: 62 as compared to the EC₅₀ for humanSIRPαV1 protein; and exhibits at least a 100-fold higher EC₅₀ for humanSIRPβ1 protein as compared to the EC₅₀ for human SIRPαV1 protein.
 33. Anantibody according to claim 31, wherein the antibody inhibits bindingbetween human SIRPα and CD47 with an IC₅₀<2.5 nM.
 34. An antibodyaccording to claim 32, wherein the antibody inhibits binding betweenhuman SIRPα and CD47 with an IC₅₀<2.5 nM.
 35. A composition comprising:an antibody or antigen binding fragment of claim 31; and apharmaceutically acceptable carrier or diluent, wherein the compositionoptionally comprises a second antibody or antigen binding fragmentthereof that induces ADCC and/or ADCP, wherein said antibody or antigenbinding fragment of claim 31 enhances the antibody-mediated destructionof cells by the second antibody.
 36. A composition comprising: anantibody or antigen binding fragment of claim 31; and a pharmaceuticallyacceptable carrier or diluent,
 37. A composition according to claim 36,further comprising a second antibody or antigen binding fragment thereofthat induces ADCC and/or ADCP.
 38. An antibody or antigen bindingfragment thereof that binds to the same epitope of human SIRPα as anantibody comprising one of the following combinations of heavy chainsequence/light chain sequence: SEQ ID NO: 78/SEQ ID NO: 90, SEQ ID NO:78/SEQ ID NO: 92, SEQ ID NO: 78/SEQ ID NO: 94, SEQ ID NO: 78/SEQ ID NO:96, SEQ ID NO: 78/SEQ ID NO: 98, SEQ ID NO: 78/SEQ ID NO: 100, SEQ IDNO: 80/SEQ ID NO: 90, SEQ ID NO: 80/SEQ ID NO: 92, SEQ ID NO: 80/SEQ IDNO: 94, SEQ ID NO: 80/SEQ ID NO: 96, SEQ ID NO: 80/SEQ ID NO: 98, SEQ IDNO: 80/SEQ ID NO: 100, SEQ ID NO: 82/SEQ ID NO: 90, SEQ ID NO: 82/SEQ IDNO: 92, SEQ ID NO: 82/SEQ ID NO: 94, SEQ ID NO: 82/SEQ ID NO: 96, SEQ IDNO: 82/SEQ ID NO: 98, SEQ ID NO: 82/SEQ ID NO: 100, SEQ ID NO: 84/SEQ IDNO: 90, SEQ ID NO: 84/SEQ ID NO: 92, SEQ ID NO: 84/SEQ ID NO: 94, SEQ IDNO: 84/SEQ ID NO: 96, SEQ ID NO: 84/SEQ ID NO: 98, SEQ ID NO: 84/SEQ IDNO: 100, SEQ ID NO: 86/SEQ ID NO: 90, SEQ ID NO: 86/SEQ ID NO: 92, SEQID NO: 86/SEQ ID NO: 94, SEQ ID NO: 86/SEQ ID NO: 96, SEQ ID NO: 86/SEQID NO: 98, SEQ ID NO: 86/SEQ ID NO: 100, SEQ ID NO: 88/SEQ ID NO: 90,SEQ ID NO: 88/SEQ ID NO: 92, SEQ ID NO: 88/SEQ ID NO: 94, SEQ ID NO:88/SEQ ID NO: 96, SEQ ID NO: 88/SEQ ID NO: 98, SEQ ID NO: 88/SEQ ID NO:100, SEQ ID NO: 10/SEQ ID NO: 20, SEQ ID NO: 10/SEQ ID NO: 22, SEQ IDNO: 10/SEQ ID NO: 24, SEQ ID NO: 10/SEQ ID NO: 26, SEQ ID NO: 10/SEQ IDNO: 28, SEQ ID NO: 12/SEQ ID NO: 20, SEQ ID NO: 12/SEQ ID NO: 22, SEQ IDNO: 12/SEQ ID NO: 24, SEQ ID NO: 12/SEQ ID NO: 26, SEQ ID NO: 12/SEQ IDNO: 28, SEQ ID NO: 14/SEQ ID NO: 20, SEQ ID NO: 14/SEQ ID NO: 22, SEQ IDNO: 14/SEQ ID NO: 24, SEQ ID NO: 14/SEQ ID NO: 26, SEQ ID NO: 14/SEQ IDNO: 28, SEQ ID NO: 16/SEQ ID NO: 20, SEQ ID NO: 16/SEQ ID NO: 22, SEQ IDNO: 16/SEQ ID NO: 24, SEQ ID NO: 16/SEQ ID NO: 26, SEQ ID NO: 16/SEQ IDNO: 28, SEQ ID NO: 18/SEQ ID NO: 20, SEQ ID NO: 18/SEQ ID NO: 22, SEQ IDNO: 18/SEQ ID NO: 24, SEQ ID NO: 18/SEQ ID NO: 26, SEQ ID NO: 18/SEQ IDNO:
 28. 39. An isolated nucleic acid encoding an antibody or antigenbinding fragment thereof that binds to human SIRPα, wherein the antibodyor antigen binding fragment comprises: a. a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO:69 or an amino acidsequence differing from SEQ ID NO: 1 by 1, 2, or 3 conservativesubstitutions, b. a heavy chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO:70 or an amino acid sequence differingfrom SEQ ID NO: 2 by 1, 2, or 3 conservative substitutions, c. a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO:71 or an amino acid sequence differing from SEQ ID NO: 3 by 1, 2, or3 conservative substitutions, d. a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO:72 or an amino acidsequence differing from SEQ ID NO: 4 by 1, 2, or 3 conservativesubstitutions, e. a light chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO:73 or an amino acid sequence differingfrom SEQ ID NO: 5 by 1, 2, or 3 conservative substitutions, and f. alight chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO:74 or an amino acid sequence differing from SEQ ID NO: 6 by 1,2, or 3 conservative substitutions. or wherein the antibody or antigenbinding fragment comprises: g. a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO:1 or an amino acidsequence differing from SEQ ID NO: 1 by 1, 2, or 3 conservativesubstitutions, h. a heavy chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO:2 or an amino acid sequence differingfrom SEQ ID NO: 2 by 1, 2, or 3 conservative substitutions, i. a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO:3 or an amino acid sequence differing from SEQ ID NO: 3 by 1, 2, or 3conservative substitutions, j. a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO:4 or an amino acidsequence differing from SEQ ID NO: 4 by 1, 2, or 3 conservativesubstitutions, k. a light chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO:5 or an amino acid sequence differingfrom SEQ ID NO: 5 by 1, 2, or 3 conservative substitutions, and l. alight chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO:6 or an amino acid sequence differing from SEQ ID NO: 6 by 1,2, or 3 conservative substitutions.
 40. An expression vector comprisingthe isolated nucleic acid of claim
 39. 41. A host cell comprisingexpression vector of claim
 40. 42. A method of producing an antibody orantigen binding fragment comprising: culturing a host cell comprising apolynucleotide of claim 39 under conditions favorable to expression ofthe polynucleotide; and optionally, recovering the antibody or antigenbinding fragment from the host cell and/or culture medium.
 43. A methodfor detecting the presence of a SIRPα peptide or a fragment thereof in asample comprising contacting the sample with an antibody or fragment anantibody or antigen binding fragment thereof that binds to human SIRPα,wherein the antibody or antigen binding fragment comprises: a. a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO:69 or an amino acid sequence differing from SEQ ID NO: 1 by 1, 2, or3 conservative substitutions, b. a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO:70 or an amino acidsequence differing from SEQ ID NO: 2 by 1, 2, or 3 conservativesubstitutions, c. a heavy chain variable region CDR3 comprising theamino acid sequence of SEQ ID NO:71 or an amino acid sequence differingfrom SEQ ID NO: 3 by 1, 2, or 3 conservative substitutions, d. a lightchain variable region CDR1 comprising the amino acid sequence of SEQ IDNO:72 or an amino acid sequence differing from SEQ ID NO: 4 by 1, 2, or3 conservative substitutions, e. a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO:73 or an amino acidsequence differing from SEQ ID NO: 5 by 1, 2, or 3 conservativesubstitutions, and f. a light chain variable region CDR3 comprising theamino acid sequence of SEQ ID NO:74 or an amino acid sequence differingfrom SEQ ID NO: 6 by 1, 2, or 3 conservative substitutions. or whereinthe antibody or antigen binding fragment comprises: g. a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:1or an amino acid sequence differing from SEQ ID NO: 1 by 1, 2, or 3conservative substitutions, h. a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO:2 or an amino acidsequence differing from SEQ ID NO: 2 by 1, 2, or 3 conservativesubstitutions, i. a heavy chain variable region CDR3 comprising theamino acid sequence of SEQ ID NO:3 or an amino acid sequence differingfrom SEQ ID NO: 3 by 1, 2, or 3 conservative substitutions, j. a lightchain variable region CDR1 comprising the amino acid sequence of SEQ IDNO:4 or an amino acid sequence differing from SEQ ID NO: 4 by 1, 2, or 3conservative substitutions, k. a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO:5 or an amino acidsequence differing from SEQ ID NO: 5 by 1, 2, or 3 conservativesubstitutions, and l. a light chain variable region CDR3 comprising theamino acid sequence of SEQ ID NO:6 or an amino acid sequence differingfrom SEQ ID NO: 6 by 1, 2, or 3 conservative substitutions; anddetecting the presence of a complex between the antibody or fragment andthe peptide; wherein detection of the complex indicates the presence ofthe SIRPα peptide.
 44. A method of treating cancer, an infection, orinfectious disease in a human subject, comprising administering to thesubject an effective amount of an antibody or antigen binding fragmentof claim
 31. 45. A method of treating cancer, an infection, orinfectious disease in a human subject, comprising administering to thesubject an effective amount of an antibody or antigen binding fragmentof claim 32.